Pesticidal genes and methods of use

ABSTRACT

Compositions having pesticidal activity and methods for their use are provided. Compositions include isolated and recombinant polypeptides having pesticidal activity, recombinant and synthetic nucleic acid molecules encoding the polypeptides, DNA constructs and vectors comprising the nucleic acid molecules, host cells comprising the vectors, and antibodies to the polypeptides. Polynucleotide sequences encoding the polypeptides can be used in DNA constructs or expression cassettes for transformation and expression in organisms of interest. The compositions and methods provided are useful for producing organisms with enhanced pest resistance or tolerance. Transgenic plants and seeds comprising a nucleotide sequence that encodes a pesticidal protein of the invention are also provided. Such plants are resistant to insects and other pests. Methods are provided for producing the various polypeptides disclosed herein, and for using those polypeptides for controlling or killing a pest. Methods and kits for detecting polypeptides of the invention in a sample are also included.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 62/270,742 filed Dec. 22, 2015 and U.S. Provisional Application Ser.No. 62/412,619 filed Oct. 25, 2016, the contents of these applicationsare herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention is drawn to methods and compositions for controllingpests, particularly plant pests.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file namedAgB024US-1029227_SeqList.txt, created on Dec. 16, 2016, and having asize of 1.18 MB. The sequence listing contained in this ASCII formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND

Pests, plant diseases, and weeds can be serious threats to crops. Lossesdue to pests and diseases have been estimated at 37% of the agriculturalproduction worldwide, with 13% due to insects, bacteria and otherorganisms.

Toxins are virulence determinants that play an important role inmicrobial pathogenicity and/or evasion of the host immune response.Toxins from the gram-positive bacterium Bacillus, particularly Bacillusthuringiensis, have been used as insecticidal proteins. Currentstrategies use the genes expressing these toxins to produce transgeniccrops. Transgenic crops expressing insecticidal protein toxins are usedto combat crop damage from insects.

While the use of Bacillus toxins has been successful in controllinginsects, resistance to Bt toxins has developed in some target pests inmany parts of the world where such toxins have been used intensively.One way of solving this problem is sowing Bt crops with alternating rowsof regular non Bt crops (refuge). An alternative method to avoid or slowdown development of insect resistance is stacking insecticidal geneswith different modes of action against insects in transgenic plants. Thecurrent strategy of using transgenic crops expressing insecticidalprotein toxins is placing increasing emphasis on the discovery of noveltoxins, beyond those already derived from the bacterium Bacillusthuringiensis. These toxins may prove useful as alternatives to thosederived from B. thuringiensis for deployment in insect- andpest-resistant transgenic plants. Thus, new toxin proteins are needed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides an amino acid alignment of SEQ ID NO: 209, 207, and 206.Highlighted regions denote regions where the amino acids are the samebetween the three polypeptides.

FIG. 2 provides the assay scoring guidelines (size×mortality matrix)employed in the western corn rootworm bioassay. “S” indicates small insize, “m” indicates medium in size, and “b” indicated big in size.

FIG. 3 provides the results of the time course assay of APG01037.1 (SEQID NO: 209) against SGSB.

FIG. 4 provides the results of the time course assay of APG01037.1 (SEQID NO: 209) against Soybean Aphids.

FIG. 5 provides the concentration-response curve of APG01037.1 (SEQ IDNO: 209) against Western Corn Rootworm.

FIG. 6 provides the results of the time course assay of APG01037.5 (SEQID NO: 211) against SGSB.

FIG. 7 shows APG1037.4-8 (SEQ ID NO: 210, 211, 212, 213, and 214) hadmortality greater than 70% mortality against Lygus.

FIG. 8 provides an alignment of SEQ ID NO: 209 against various activevariants. The sequences present in the alignment are as follows:APG01037.1 (SEQ ID NO: 209); APG01037.0 (SEQ ID NO: 208); APG01037.4(SEQ ID NO: 210); APG01037.5 (SEQ ID NO: 211); APG01037.6 (SEQ ID NO:212); APG01037.7 (SEQ ID NO: 213); APG01037.8 (SEQ ID NO: 214);APG00556.0 (SEQ ID NO: 205); APG00556.1 (SEQ ID NO: 206); and APG00623.0(SEQ ID NO: 207).

FIG. 9 provides the sequences identity relationships of various activevariants of SEQ ID NO: 209.

SUMMARY

Compositions having pesticidal activity and methods for their use areprovided. Compositions include isolated and recombinant polypeptidesequences having pesticidal activity, recombinant and synthetic nucleicacid molecules encoding the pesticidal polypeptides, DNA constructscomprising the nucleic acid molecules, vectors comprising the nucleicacid molecules, host cells comprising the vectors, and antibodies to thepesticidal polypeptides. Nucleotide sequences encoding the polypeptidesprovided herein can be used in DNA constructs or expression cassettesfor transformation and expression in organisms of interest, includingmicroorganisms and plants.

The compositions and methods provided herein are useful for theproduction of organisms with enhanced pest resistance or tolerance.These organisms and compositions comprising the organisms are desirablefor agricultural purposes. Transgenic plants and seeds comprising anucleotide sequence that encodes a pesticidal protein of the inventionare also provided. Such plants are resistant to insects and other pests.

Methods are provided for producing the various polypeptides disclosedherein, and for using those polypeptides for controlling or killing apest. Methods and kits for detecting polypeptides of the invention in asample are also included.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

I. Polynucleotides and Polypeptides

Compositions and method for conferring pesticidal activity to anorganism are provided. The modified organism exhibits pesticidalresistance or tolerance. Recombinant pesticidal proteins, orpolypeptides and fragments and variants thereof that retain pesticidalactivity, are provided and include those set forth in SEQ ID NOs: 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, and/or 218. Thepesticidal proteins are biologically active (e.g., pesticidal) againstpests including insects, fungi, nematodes, and the like. Nucleotidesencoding the pesticidal polypeptides, including for example, SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, and/or 218 oractive fragments or variants thereof, can be used to produce transgenicorganisms, such as plants and microorganisms. The pesticidal proteinsare biologically active (for example, are pesticidal) against pestsincluding insects, fungi, nematodes, and the like. Polynucleotidesencoding the pesticidal polypeptides, including for example, SEQ ID NOS:1-218 or active fragments or variants thereof, can be used to producetransgenic organisms, such as plants and microorganisms. The transformedorganisms are characterized by genomes that comprise at least one stablyincorporated DNA construct comprising a coding sequence for a pesticidalprotein disclosed herein. In some embodiments, the coding sequence isoperably linked to a promoter that drives expression of the encodedpesticidal polypeptide. Accordingly, transformed microorganisms, plantcells, plant tissues, plants, seeds, and plant parts are provided. Asummary of various polypeptides, active variants and fragments thereof,and polynucleotides encoding the same are set forth below in Table 1. Asnoted in Table 1, various forms of polypeptides are provided. Fulllength pesticidal polypeptides, as well as, modified versions of theoriginal full-length sequence (i.e., variants) are provided. Table 1further denotes “CryBP1” sequences. Such sequences (SEQ ID NOS: 103 and178) comprise accessory polypeptides that can be associated with some ofthe toxin genes. In such instances, the CryBP1 sequences can be usedalone or in combination with any of the pesticidal polypeptides providedherein. Table 1 further provides Split-Cry C-terminus polypeptides (SEQID NO: 30, 112, 135, 193, or 203). Such sequence comprise the sequenceof a downstream protein that has homology to the C-terminal end of theCry class of toxin genes and are usually found after a Cry gene that isnot full-length and is missing the expected C-terminal region.

TABLE 1 Summary of SEQ ID NOs, Gene Class, and Variants thereof Full-CryBP1 length Modified SEQ Split-Cry C- Similarity SEQ SEQ ID IDterminus SEQ Gene Identity from nearest from nearest Gene Name ID No.No.(s) No. ID No. Class non-APG non-APG Homologs APG00326 1 2, 3 Cry4A85, 90, 95, 96, 97, 98, 99 90, 95, 96, 97, 98, Cry4Aa2 (80.48% 99identity, 86.85% similarity) ABM97547.1 (79.57% identity, 86.35%similarity) ABR12216.1 (79.24% identity, 86.02% similarity) A9XI31_BACTU(79.16% identity, 85.94% similarity) APG00343 4 5 Cry 60, 65, 70, 75,80, 85, 90, 75, 80, 85, 90, 95, APG00514 (89.36% 95, 96, 97, 98, 99 96,97, 98, 99 identity, 94.53% similarity) APG00654 (80.12% identity,87.72% similarity) APG00164 (77.19% identity, 84.36% similarity)CBL59393.1 (56.49% identity, 72.61% similarity) CBL59396.1 (56.49%identity, 72.47% similarity) D5H3I8_BACTG (56.35% identity, 72.47%similarity) APG00084 (52.79% identity, 69.81% similarity) Cry8Aa1(35.34% identity, 43.05% similarity) APG00383 6 7 Cry 40, 45, 50, 55,60, 65, 70, 55, 60, 65, 70, 75, APG00101 (96.93% 75, 80, 85, 90, 95, 96,97, 80, 85, 90, 95, 96, identity, 97.16% 98, 99 97, 98, 99 similarity)APG00034 (61.93% identity, 74.85% similarity) APG00048 (52.09% identity,63.77% similarity) US_2011_0231963_A1- 9 (37.27% identity, 51.39%similarity) Cry13Aa1 (36.96% identity, 53.97% similarity) APG00493 8 9Mtx 90, 95, 96, 97, 98, 99 90, 95, 96, 97, 98, WP_020294695.1 99 (85.08%identity, 89.15% similarity) US20140007292A1_4 (84.41% identity, 88.81%similarity) US20140007292A1_2 (82.71% identity, 88.14% similarity)US20140007292A1_332 (76.61% identity, 83.39% similarity) APG00659(72.26% identity, 80.0% similarity) APG00494 10 11 Bin 90, 95, 96, 97,98, 99 95, 96, 97, 98, 99 APG00731 (89.01% identity, 93.03% similarity)APG00669 (88.2% identity, 92.49% similarity) APG00035 (87.67% identity,92.76% similarity) APG00568 (86.86% identity, 91.96% similarity)WP_000143307.1 (86.6% identity, 91.69% similarity) APG00356 (86.6%identity, 91.96% similarity) APG00284 (86.06% identity, 92.23%similarity) WP_000143308.1 (85.79% identity, 91.69% similarity)WP_050845516.1 (84.18% identity, 90.35% similarity) APG00735 (75.19%identity, 82.78% similarity) Cry35Ac2 (22.22% identity, 41.06%similarity) APG00495 12 13 Mtx 80, 85, 90, 95, 96, 97, 98, 85, 90, 95,96, 97, WP_016099228.1 99 98, 99 (75.77% identity, 82.52% similarity)APG00693 (70.06% identity, 79.64% similarity) AGP17978.1 (68.77%identity, 78.98% similarity) APG00049 (50.76% identity, 62.69%similarity) EEM56710.1 (50.46% identity, 64.74% similarity)WP_008180054.1 (50.3% identity, 63.94% similarity) APG00513 14 Mtx 65,70, 75, 80, 85, 90, 95, 80, 85, 90, 95, 96, APG00846 (85.63% 96, 97, 98,99 97, 98, 99 identity, 91.38% similarity) AGA40030.1 (62.71% identity,77.12% similarity) APG00609 (53.44% identity, 69.15% similarity)APG00224 (53.33% identity, 69.44% similarity) CAA67205.1 (43.06%identity, 60.06% similarity) AGA40032.1 (40.93% identity, 53.85%similarity) R8R7A7_BACCE (20.75% identity, 31.6% similarity) APG00514 1516 Cry 60, 65, 70, 75, 80, 85, 90, 75, 80, 85, 90, 95, APG00343 (89.36%95, 96, 97, 98, 99 96, 97, 98, 99 identity, 94.53% similarity) APG00654(81.47% identity, 88.38% similarity) APG00164 (78.09% identity, 85.29%similarity) CBL59393.1 (56.7% identity, 72.36% similarity) CBL59396.1(56.55% identity, 72.36% similarity) D5H3I8_BACTG (56.55% identity,72.22% similarity) APG00084 (52.7% identity, 69.18% similarity) Cry8Aa1(36.45% identity, 43.86% similarity) APG00524 17 18, 19 Cry14 55, 60,65, 70, 75, 80, 85, 70, 75, 80, 85, 90, APG00651 (77.43% 90, 95, 96, 97,98, 99 95, 96, 97, 98, 99 identity, 84.21% similarity) Cry14Aa1 (54.15%identity, 67.51% similarity) APG00052 (52.77% identity, 65.77%similarity) Cry21Ba1 (45.51% identity, 61.62% similarity) Cry21Ca2(43.99% identity, 60.21% similarity) APG00528 20 Mtx 55, 60, 65, 70, 75,80, 85, 65, 70, 75, 80, 85, APG00661 (57.14% 90, 95, 96, 97, 98, 99 90,95, 96, 97, 98, identity, 66.98% 99 similarity) US_2008_0070829_A1- 25(54.57% identity, 64.87% similarity) AGA40029.1 (53.41% identity, 62.82%similarity) CAA63374.1 (41.55% identity, 51.37% similarity) AGA40031.1(41.41% identity, 54.12% similarity) APG00533 21 Cry11B 80, 85, 90, 95,96, 97, 98, 85, 90, 95, 96, 97, APG00607 (97.89% 99 98, 99 identity,97.89% similarity) Cry11Bb1 (77.26% identity, 84.18% similarity)ADI59541.1 (74.56% identity, 80.60% similarity) Cry11Ba1 (74.13%identity, 82.44% similarity) AEK06469.1 (67.07% identity, 73.79%similarity) APG00534 22 23 Cry 50, 55, 60, 65, 70, 75, 80, 65, 70, 75,80, 85, APG00120 (82.82% 85, 90, 95, 96, 97, 98, 99 90, 95, 96, 97, 98,identity, 88.17% 99 similarity) WP_001070417.1 (45.9% identity, 60.78%similarity) Cry41Ba2 (44.35% identity, 59.13% similarity) Cry41Aa1(41.39% identity, 56.51% similarity) Cry41Ab1 (38.61% identity, 54.79%similarity) APG00536 24 25, 26 Cry 85, 90, 95, 96, 97, 98, 99 90, 95,96, 97, 98, WO_2014_138339-52 99 (83.47% identity, 88.74% similarity)APG00046 (83.47% identity, 88.74% similarity) WO_2014_138339-53 (83.35%identity, 88.5% similarity) WO_2014_138339-54 (76.65% identity, 80.24%similarity) Cry68Aa1 (33.02% identity, 46.38% similarity) APG00537 2728, 29 30 Cry53A 85, 90, 95, 96, 97, 98, 99 90, 95, 96, 97, 98,AGP17986.1 (83.11% 99 identity, 87.81% similarity) WP_014990538.1 (80.7%identity, 86.26% similarity) ACP43734.1 (80.26% identity, 85.96%similarity) APG00606 (77.73% identity, 82.82% similarity) Cry53Aa1(77.29% identity, 83.41% similarity) APG00543 31 32 Cry27A 90, 95, 96,97, 98, 99 95, 96, 97, 98, 99 WP_016098322.1 (89.92% identity, 94.26%similarity) AGV55018.1 (86.13% identity, 89.02% similarity) Cry27Aa1(75.99% identity, 83.43% similarity) WO_2014_102697-1 (40.07% identity,53.93% similarity) WO_2014_102697-2 (39.83% identity, 53.39% similarity)APG00555 33 34, 35 Cry 75, 80, 85, 90, 95, 96, 97, 85, 90, 95, 96, 97,CA_2843744-20 98, 99 98, 99 (71.12% identity, 80.7% similarity)CA_2843744-22 (69.81% identity, 79.1% similarity) APG01028 (66.91%identity, 76.83% similarity) US20130227743A1_26 (59.91% identity, 73.13%similarity) APG00039 (50.93% identity, 63.04% similarity) Cry53Aa1(39.83% identity, 55.76% similarity) APG00557 36 37 Bin 30, 35, 40, 45,50, 55, 60, 45, 50, 55, 60, 65, APG00050 (60.99% 65, 70, 75, 80, 85, 90,95, 70, 75, 80, 85, 90, identity, 71.39% 96, 97, 98, 99 95, 96, 97, 98,99 similarity) APG00461 (46.58% identity, 59.62% similarity) APG00474(43.05% identity, 53.67% similarity) APG00629 (42.52% identity, 56.78%similarity) Cry49Aa1 (29.92% identity, 42.37% similarity) APG00558 3839, 40 Cry 45, 50, 55, 60, 65, 70, 75, 60, 65, 70, 75, 80, AGV55021.1(42.52% 80, 85, 90, 95, 96, 97, 98, 85, 90, 95, 96, 97, identity, 56.5%99 98, 99 similarity) APG00153 (36.59% identity, 50.49% similarity)Cry53Aa1 (33.51% identity, 50.0% similarity) APG00565 41 42, 43 Cry30D90, 95, 96, 97, 98, 99 95, 96, 97, 98, 99 Cry30Db1 (88.73% identity,90.87% similarity) Cry30Da1 (86.31% identity, 89.87% similarity)Cry30Ga2 (70.54% identity, 78.19% similarity) Cry30Ga1 (69.97% identity,77.76% similarity) ADK47393.1 (69.96% identity, 77.86% similarity)APG00972 (63.5% identity, 75.94% similarity) APG00566 44 45 Mtx 75, 80,85, 90, 95, 96, 97, 85, 90, 95, 96, 97, APG01086 (97.39% 98, 99 98, 99identity, 99.02% similarity) APG00201 (82.03% identity, 91.5%similarity) APG00006 (80.07% identity, 90.52% similarity) APG00260(78.43% identity, 88.24% similarity) APG00036 (75.24% identity, 84.04%similarity) APG00022 (75.16% identity, 83.33% similarity) WP_000963933.1(73.62% identity, 84.04% similarity) US20130227743A1_100 (72.22%identity, 83.33% similarity) US20130227743A1_60 (45.1% identity, 50.33%similarity) ABV97497.1 (24.15% identity, 39.49% similarity) APG00572 46PI-PLC 40, 45, 50, 55, 60, 65, 70, 55, 60, 65, 70, 75, WP_016084067.175, 80, 85, 90, 95, 96, 97, 80, 85, 90, 95, 96, (38.65% identity, 98, 9997, 98, 99 50.15% similarity) WP_000836979.1 (38.12% identity, 49.85%similarity) EEM93103.1 (37.91% identity, 49.05% similarity) Cry73Aa(17.75% identity, 29.44% similarity) APG00587 47 48 Cry 50, 55, 60, 65,70, 75, 80, 65, 70, 75, 80, 85, WP_048536363.1 85, 90, 95, 96, 97, 98,99 90, 95, 96, 97, 98, (45.21% identity, 99 60.54% similarity)WP_048536362.1 (31.16% identity, 46.29% similarity) US20130227743A1_200(27.52% identity, 44.36% similarity) Cry42Aa1 (14.96% identity, 24.9%similarity) APG00939 49 50 Vip3 95, 96, 97, 98, 99 97, 98, 99WP_048517127.1 (94.07% identity, 96.88% similarity) APG00077 (81.86%identity, 88.35% similarity) APG00278 (81.14% identity, 86.94%similarity) APG00875 (80.82% identity, 86.19% similarity) APG00173(80.0% identity, 86.19% similarity) APG00175 (80.0% identity, 86.6%similarity) APG00657 (73.63% identity, 82.73% similarity) APG01003(72.64% identity, 80.23% similarity) WP_050001316.1 (67.11% identity,78.07% similarity) CA_2866166-1528 (23.27% identity, 39.55% similarity)Vip3Aa18 (23.21% identity, 40.48% similarity) APG00606 51 52, 53 Cry5380, 85, 90, 95, 96, 97, 98, 85, 90, 95, 96, 97, APG00537 (77.73% 99 98,99 identity, 82.82% similarity) WP_014990538.1 (76.17% identity, 82.6%similarity) ACP43734.1 (76.02% identity, 82.6% similarity) AGP17986.1(75.98% identity, 82.53% similarity) Cry53Aa1 (71.93% identity, 79.68%similarity) APG01028 (54.05% identity, 66.71% similarity) APG00607 54Cry11B 80, 85, 90, 95, 96, 97, 98, 85, 90, 95, 96, 97, APG00533 (97.89%99 98, 99 identity, 97.89% similarity) Cry11Bb1 (77.49% identity, 83.77%similarity) APG00608 55 56 Cry 40, 45, 50, 55, 60, 65, 70, 55, 60, 65,70, 75, APG00034 (49.72% 75, 80, 85, 90, 95, 96, 97, 80, 85, 90, 95, 96,identity, 62.43% 98, 99 97, 98, 99 similarity) APG00002 (44.42%identity, 56.00% similarity) APG00383 (43.43% identity, 58.13%similarity) APG00101 (42.70% identity, 56.97% similarity) APG00048(40.55% identity, 54.86% similarity) Cry13Aa1 (38.05% identity, 53.68%similarity) APG00609 57 Mtx 65, 70, 75, 80, 85, 90, 95, 75, 80, 85, 90,95, APG00224 (63.1% 96, 97, 98, 99 96, 97, 98, 99 identity, 73.52%similarity) AGA40030.1 (60.72% identity, 71.87% similarity) APG00513(53.44% identity, 69.15% similarity) APG00846 (52.62% identity, 67.49%similarity) AGA40032.1 (39.61% identity, 54.21% similarity) CAA67205.1(39.6% identity, 57.83% similarity) C4B693_CLOBO (20.11% identity,35.92% similarity) APG00622 58 59 Cry5 60, 65, 70, 75, 80, 85, 90, 70,75, 80, 85, 90, Cry5Ba1 (56.24% 95, 96, 97, 98, 99 95, 96, 97, 98, 99identity, 65.33% similarity) Cry5Ba2 (56.24% identity, 65.27%similarity) Cry5Ba3 (56.03% identity, 65.13% similarity) APG00217(53.75% identity, 62.25% similarity) APG00624 60 61 Cyt 45, 50, 55, 60,65, 70, 75, 60, 65, 70, 75, 80, Cyt1Da1 (43.72% 80, 85, 90, 95, 96, 97,98, 85, 90, 95, 96, 97, identity, 58.52% 99 98, 99 similarity)WP_000079177.1 (42.96% identity, 60.14% similarity) WP_043939324.1(42.42% identity, 59.28% similarity) APG00637 62 63, 64 Cry 60, 65, 70,75, 80, 85, 90, 70, 75, 80, 85, 90, APG00115 (92.62% 95, 96, 97, 98, 9995, 96, 97, 98, 99 identity, 94.99% similarity) US_2015_0218583_A1- 3(57.11% identity, 65.14% similarity) EJR93120.1 (56.83% identity, 67.66%similarity) AGP18037.1 (54.79% identity, 64.79% similarity) Cry32Ea1(36.19% identity, 48.14% similarity) APG00638 65 PI-PLC 80, 85, 90, 95,96, 97, 98, 90, 95, 96, 97, 98, WP_050845433.1 99 99 (79.88% identity,86.65% similarity) AGA40046.1 (68.23% identity, 79.34% similarity)WP_000513490.1 (67.84% identity, 78.36% similarity) Cyt1Da1 (22.69%identity, 34.85% similarity) APG00641 66 67 Mtx 35, 40, 45, 50, 55, 60,65, 50, 55, 60, 65, 70, APG00807 (73.68% 70, 75, 80, 85, 90, 95, 96, 75,80, 85, 90, 95, identity, 82.57% 97, 98, 99 96, 97, 98, 99 similarity)APG00434 (49.68% identity, 66.77% similarity) WP_054770413.1 (31.91%identity, 47.42% similarity) Cry64Aa1 (31.68% identity, 47.2%similarity) APG00643 68 69 Cry 85, 90, 95, 96, 97, 98, 99 90, 95, 96,97, 98, APG00800 (87.98% 99 identity, 93.99% similarity) WP_012259841.1(80.77% identity, 88.77% similarity) US20130227743A1_24 (78.12%identity, 84.75% similarity) APG00088 (64.81% identity, 75.19%similarity) WP_025988975.1 (62.92% identity, 67.17% similarity) Cry8Ca1(24.72% identity, 34.83% similarity) APG00644 70 71, 72 Cry 60, 65, 70,75, 80, 85, 90, 65, 70, 75, 80, 85, US20130227743A1_202 95, 96, 97, 98,99 90, 95, 96, 97, 98, (58.97% identity, 99 63.99% similarity)WP_048536362.1 (20.86% identity, 34.16% similarity) AFU11621.1 (19.84%identity, 32.76% similarity) US20130227743A1_204 (19.27% identity,32.47% similarity) APG00648 73 74 Bin 70, 75, 80, 85, 90, 95, 96, 80,85, 90, 95, 96, APG00261 (69.58% 97, 98, 99 97, 98, 99 identity, 79.05%similarity) APG00988 (67.83% identity, 77.06% similarity) WP_001258160.1(66.17% identity, 78.7% similarity) WP_001258161.1 (66.17% identity,78.7% similarity) US20130227743A1_10 (66.0% identity, 78.5% similarity)APG00223 (61.22% identity, 71.43% similarity) APG00454 (61.22% identity,71.43% similarity) APG00242 (61.0% identity, 71.2% similarity) APG00335(60.77% identity, 71.2% similarity) APG00724 (55.9% identity, 68.19%similarity) APG00716 (55.09% identity, 69.23% similarity) APG00806(52.8% identity, 64.96% similarity) Cry35Ab4 (22.58% identity, 38.02%similarity) APG00649 75 76 Bin 70, 75, 80, 85, 90, 95, 96, 85, 90, 95,96, 97, APG00413 (69.63% 97, 98, 99 98, 99 identity, 81.68% similarity)APG00230 (68.23% identity, 81.25% similarity) WP_017154552.1 (67.72%identity, 80.31% similarity) WP_050001305.1 (67.62% identity, 79.9%similarity) US_2014_0033363_A1- 2 (66.06% identity, 78.76% similarity)APG00596 (65.28% identity, 77.98% similarity) APG00373 (65.27% identity,77.02% similarity) APG00090 (63.45% identity, 78.85% similarity)Cry35Ab2 (22.78% identity, 34.17% similarity) APG00651 77 78 Cry14 60,65, 70, 75, 80, 85, 90, 75, 80, 85, 90, 95, APG00524 (77.43% 95, 96, 97,98, 99 96, 97, 98, 99 identity, 84.21% similarity) Cry14Aa1 (57.4%identity, 70.63% similarity) APG00052 (54.52% identity, 68.23%similarity) APG00657 79 80 Vip3 75, 80, 85, 90, 95, 96, 97, 85, 90, 95,96, 97, APG00175 (74.79% 98, 99 98, 99 identity, 82.85% similarity)APG00939 (73.63% identity, 82.73% similarity) APG00077 (73.33% identity,81.8% similarity) WP_048517127.1 (73.21% identity, 82.55% similarity)APG00273 (72.12% identity, 81.66% similarity) APG00278 (71.29% identity,80.48% similarity) APG00173 (71.03% identity, 80.48% similarity)APG01003 (67.97% identity, 78.06% similarity) WP_050001316.1 (66.7%identity, 78.35% similarity) Vip3Aa13 (23.92% identity, 39.9%similarity) APG00659 81 82 Mtx 80, 85, 90, 95, 96, 97, 98, 85, 90, 95,96, 97 US20140007292A1_332 99 98, 99 (79.39% identity, 84.46%similarity) CA_2878263-105 (78.04% identity, 84.8% similarity)US20140007292A1_2 (76.01% identity, 83.11% similarity) WP_020294695.1(75.68% identity, 83.78% similarity) APG00493 (72.26% identity, 80.0%similarity) APG00661 83 Mtx 85, 90, 95, 96, 97, 98, 99 95, 96, 97, 98,99 APG06508 (96.41% identity, 97.31% similarity) APG09801 (94.91%identity, 97.01% similarity) US_2008_0070829_A1- 25 (83.63% identity,90.48% similarity) AGA40029.1 (74.63% identity, 84.78% similarity)CAA63374.1 (58.4% identity, 68.09% similarity) APG00528 (57.14%identity, 66.98% similarity) AGA40031.1 (46.57% identity, 63.58%similarity) APG00662 84 85, 86 Cry69 75, 80, 85, 90, 95, 96, 97, 85, 90,95, 96, 97, APG00079 (77.58% 98, 99 98, 99 identity, 85.14% similarity)CA_2753918-13 (72.5% identity, 80.38% similarity) US_2011_0197314_A1- 13(72.5% identity, 80.3% similarity) APG00786 (70.46% identity, 77.45%similarity) AFU17214.1 (66.67% identity, 76.58% similarity) APG00059(65.69% identity, 75.59% similarity) Cry69Aa1 (63.54% identity, 73.38%similarity) APG00663 87 88 Cry5 50, 55, 60, 65, 70, 75, 80, 65, 70, 75,80, 85, APG00217 (51.48% 85, 90, 95, 96, 97, 98, 99 90, 95, 96, 97, 98,identity, 62.91% 99 similarity) Cry5Ca2 (49.46% identity, 61.2%similarity) Cry5Ba1 (49.27% identity, 60.66% similarity) Cry5Ba3 (49.16%identity, 60.75% similarity) Cry5Ba2 (49.12% identity, 60.58%similarity) APG00664 89 90 Cry54 65, 70, 75, 80, 85, 90, 95, 75, 80, 85,90, 95, AGA40050.1 (60.55% 96, 97, 98, 99 96, 97, 98, 99 identity,72.55% similarity) Cry54Aa2 (59.72% identity, 71.59% similarity)APG00864 (58.9% identity, 70.48% similarity) Cry54Aa1 (56.40% identity,68.39% similarity) AFU17297.1 (55.65% identity, 66.80% similarity)APG00672 91 92 Cry 95, 96, 97, 98, 99 96, 97, 98, 99 AGA40064.1 (91.5%identity, 95.46% similarity) APG00045 (83.92% identity, 90.26%similarity) APG00110 (61.34% identity, 73.07% similarity) WP_016110336.1(49.54% identity, 63.7% similarity) AGP18054.1 (31.23% identity, 47.85%similarity) Cry70Ba1 (23.84% identity, 40.99% similarity) APG00673 9394, 95 Cry32 70, 75, 80, 85, 90, 95, 96, 80, 85, 90, 95, 96, Cry32Ea1(66.69% 97, 98, 99 97, 98, 99 identity, 75.3% similarity) AGU13851.1(66.69% identity, 75.19% similarity) APG00054 (59.06% identity, 69.02%similarity) APG00310 (57.34% identity, 67.43% similarity) APG00068(56.63% identity, 68.54% similarity) APG00469 (54.97% identity, 66.89%similarity) APG00710 (51.05% identity, 61.25% similarity) APG00687(50.5% identity, 61.08% similarity) APG00674 96 Bin 40, 45, 50, 55, 60,65, 70, 55, 60, 65, 70, 75, APG00472 (71.06% 75, 80, 85, 90, 95, 96, 97,80, 85, 90, 95, 96, identity, 83.67% 98, 99 97, 98, 99 similarity)APG00642 (69.71% identity, 80.86% similarity) WP_029439068.1 (35.26%identity, 50.96% similarity) WP_002187944.1 (31.71% identity, 47.56%similarity) WP_028595059.1 (31.41% identity, 47.49% similarity) Cry35Aa2(24.76% identity, 40.0% similarity) APG00675 97 Vip3 35, 40, 45, 50, 55,60, 65, 45, 50, 55, 60, 65, APG00131 (62.46% 70, 75, 80, 85, 90, 95, 96,70, 75, 80, 85, 90, identity, 78.65% 97, 98, 99 95, 96, 97, 98, 99similarity) APG00181 (60.35% identity, 74.87% similarity) APG00038(60.35% identity, 74.26% similarity) Vip3Ba1 (30.77% identity, 44.23%similarity) APG00677 98 Cry 40, 45, 50, 55, 60, 65, 70, 55, 60, 65, 70,75, APG00003 (65.55% 75, 80, 85, 90, 95, 96, 97, 80, 85, 90, 95, 96,identity, 77.44% 98, 99 97, 98, 99 similarity) BAE79727.1 (35.46%identity, 51.52% similarity) KIQ78015.1 (34.63% identity, 50.13%similarity) US20130227743A1_74 (34.18% identity, 49.67% similarity)Cry4Aa1 (21.23% identity, 30.85% similarity) APG00679 99 100 Cry1D 80,85, 90, 95, 96, 97, 98, 90, 95, 96, 97, 98, Cry1Db1 (77.55% 99 99identity, 85.86% similarity) Cry1Da1 (77.52% identity, 84.87%similarity) Cry1Db2 (77.46% identity, 85.78% similarity) AEH31432.1(77.35% identity, 85.04% similarity) WP_000405159.1 (77.35% identity,85.13% similarity) APG00687 101 102 103 Cry32C 90, 95, 96, 97, 98, 9995, 96, 97, 98, 99 AGU13873.1 (85.7% identity, 90.23% similarity)Cry32Ca1 (84.22% identity, 89.92% similarity) APG00710 (55.17% identity,65.22% similarity) APG00430 (54.82% identity, 64.77% similarity)APG00469 (53.5% identity, 64.89% similarity) APG00056 (52.93% identity,65.09% similarity) APG00058 (50.85% identity, 63.43% similarity)APG00105 (50.75% identity, 62.69% similarity) APG00673 (50.5% identity,61.08% similarity) APG00688 104 105 Cry 40, 45, 50, 55, 60, 65, 70, 45,50, 55, 60, 65, AGU13850.1 (36.79% 75, 80, 85, 90, 95, 96, 97, 70, 75,80, 85, 90, identity, 44.66% 98, 99 95, 96, 97, 98, 99 similarity)AGU13871.1 (34.44% identity, 43.46% similarity) AGU13875.1 (32.76%identity, 40.49% similarity) Cry32Ea1 (29.64% identity, 40.09%similarity) APG00693 106 107, 108 Mtx 85, 90, 95, 96, 97, 98, 99 90, 95,96, 97, 98, AGP17978.1 (81.23% 99 identity, 88.0% similarity)WP_016099228.1 (80.62% identity, 86.46% similarity) APG00495 (70.06%identity, 79.64% similarity) EEM56710.1 (50.46% identity, 66.57%similarity) WP_008180054.1 (50.0% identity, 65.85% similarity) APG00695109 110, 111 112 Cry40D 80, 85, 90, 95, 96, 97, 98, 90, 95, 96, 97, 98,Cry40Da1 (79.91% 99 99 identity, 86.06% similarity) APG00204 (61.61%identity, 74.7% similarity) APG00111 (52.52% identity, 61.84%similarity) APG00701 113 114 Bin 70, 75, 80, 85, 90, 95, 96, 85, 90, 95,96, 97, WP_002191947.1 97, 98, 99 98, 99 (69.07% identity, 81.44%similarity) APG00243 (68.99% identity, 80.36% similarity) WP_000839920.1(68.86% identity, 80.51% similarity) WP_002166959.1 (68.81% identity,81.44% similarity) APG00065 (66.91% identity, 77.94% similarity)APG00459 (66.1% identity, 77.8% similarity) APG00412 (65.83% identity,77.64% similarity) APG00132 (65.6% identity, 78.87% similarity) APG00806(64.75% identity, 77.75% similarity) APG00716 (63.5% identity, 77.89%similarity) APG00724 (63.05% identity, 74.88% similarity) APG00988(61.32% identity, 75.06% similarity) Cry35Ab3 (23.53% identity, 38.82%similarity) APG00702 115 116 Cry 65, 70, 75, 80, 85, 90, 95, 75, 80, 85,90, 95, WP_048536362.1 96, 97, 98, 99 96, 97, 98, 99 (62.74% identity,70.16% similarity) APG00401 (60.03% identity, 71.78% similarity)APG00255 (59.87% identity, 71.62% similarity) WP_048536324.1 (43.52%identity, 56.17% similarity) WP_048536363.1 (33.39% identity, 49.37%similarity) Cry73Aa (19.2% identity, 29.02% similarity) APG00703 117118, 119 Cry 80, 85, 90, 95, 96, 97, 98, 85, 90, 95, 96, 97, AGP18005.1(77.0% 99 98, 99 identity, 84.57% similarity) AGA40033.1 (51.02%identity, 64.04% similarity) AEH76823.1 (49.6% identity, 61.56%similarity) Cry21Ca2 (40.2% identity, 55.84% similarity) APG00705 120121, 122, Cry70B 95, 96, 97, 98, 99 96, 97, 98, 99 ETT82181.1 (91.91%123, 124, identity, 95.59% 125 similarity) Cry70Bb1 (91.67% identity,95.71% similarity) WP_016093954.1 (91.67% identity, 95.34% similarity)APG00526 (86.9% identity, 92.53% similarity) APG00025 (84.82% identity,91.55% similarity) APG00728 (84.09% identity, 90.94% similarity)APG00595 (65.25% identity, 77.64% similarity) APG00027 (57.19% identity,72.54% similarity) APG00706 126 Bin 40, 45, 50, 55, 60, 65, 70, 55, 60,65, 70, 75, APG00063 (39.49% 75, 80, 85, 90, 95, 96, 97, 80, 85, 90, 95,96, identity, 51.87% 98, 99 97, 98, 99 similarity) Cry49Ab1 (39.13%identity, 52.96% similarity) APG00707 127 128 Mtx 60, 65, 70, 75, 80,85, 90, 75, 80, 85, 90, 95, APG00939 (55.76% 95, 96, 97, 98, 99 96, 97,98, 99 identity, 68.48% similarity) AGA40045.1 (55.49% identity, 71.04%similarity) APG00146 (50.47% identity, 66.36% similarity) APG00351(50.14% identity, 65.13% similarity) WP_000794514.1 (49.84% identity,67.29% similarity) US20130227743A1_102 (46.27% identity, 62.11%similarity) ETK27180.1 (41.61% identity, 56.52% similarity) APG00710 129130, 131 Cry32 95, 96, 97, 98, 99 95, 96, 97, 98, 99 AGU13855.1 (91.28%identity, 94.34% similarity) US20110203014_23 (89.51% identity, 92.47%similarity) AGU13869.1 (89.43% identity, 92.47% similarity) APG00430(78.11% identity, 83.98% similarity) APG00056 (69.24% identity, 79.31%similarity) APG00058 (66.26% identity, 75.28% similarity) APG00687(55.17% identity, 65.22% similarity) Cry32Ab1 (53.65% identity, 64.62%similarity) APG00469 (53.09% identity, 64.73% similarity) APG00504(52.35% identity, 64.28% similarity) APG00673 (51.24% identity, 61.54%similarity) APG00718 132 Cry 40, 45, 50, 55, 60, 65, 70, 60, 65, 70, 75,80, APG00860 (54.49% 75, 80, 85, 90, 95, 96, 97, 85, 90, 95, 96, 97,identity, 73.08% 98, 99 98, 99 similarity) AGA40044.1 (38.97% identity,55.59% similarity) APG00721 133 134 135 Cry65 55, 60, 65, 70, 75, 80,85, 65, 70, 75, 80, 85, APG00136 (95.85% 90, 95, 96, 97, 98, 99 90, 95,96, 97, 98, identity, 97.36% 99 similarity) Cry65Aa2 (51.84% identity,60.91% similarity) APG00123 (38.24% identity, 51.05% similarity)APG00722 136 Bin 70, 75, 80, 85, 90, 95, 96, 80, 85, 90, 95, 96,AGP18023.1 (67.35% 97, 98, 99 97, 98, 99 identity, 78.52% similarity)APG00340 (65.84% identity, 76.4% similarity) APG00151 (65.25% identity,76.01% similarity) US20130227743A1_40 (65.12% identity, 72.03%similarity) US20130227743A1_48 (36.16% identity, 45.72% similarity)Cry4Ba4 (25.42% identity, 34.99% similarity) APG00724 137 138 Bin 70,75, 80, 85, 90, 95, 96, 80, 85, 90, 95, 96, APG00407 (88.61% 97, 98, 9997, 98, 99 identity, 94.31% similarity) APG00385 (81.89% identity,88.83% similarity) APG00419 (78.47% identity, 84.65% similarity)APG00229 (77.91% identity, 83.01% similarity) APG00767 (73.18% identity,82.82% similarity) APG00716 (73.15% identity, 81.03% similarity)WP_002166959.1 (68.46% identity, 77.51% similarity) WP_002191947.1(68.22% identity, 77.51% similarity) WP_000839920.1 (68.06% identity,79.12% similarity) APG00806 (67.08% identity, 79.7% similarity) APG00701(63.05% identity, 74.88% similarity) APG00988 (60.78% identity, 71.32%similarity) Cry35Ac1 (21.46% identity, 36.91% similarity) APG00726 139140, 141 Cry 75, 80, 85, 90, 95, 96, 97, 85, 90, 95, 96, 97,CA_2843744-9 98, 99 98, 99 (71.01% identity, 81.16% similarity)CA_2843744-7 (68.68% identity, 78.64% similarity) US20130227743A1_74(36.45% identity, 51.31% similarity) Cry54Ba1 (34.34% identity, 47.11%similarity) APG00729 142 143 Bin 35, 40, 45, 50, 55, 60, 65, 50, 55, 60,65, 70, WP_048517129.1 70, 75, 80, 85, 90, 95, 96, 75, 80, 85, 90, 95,(30.75% identity, 97, 98, 99 96, 97, 98, 99 47.94% similarity)WP_048548696.1 (30.37% identity, 46.03% similarity) WP_002090518.1(29.98% identity, 47.96% similarity) Cry35Ab5 (24.7% identity, 38.37%similarity) APG00735 144 145 Bin 85, 90, 95, 96, 97, 98, 99 90, 95, 96,97, 98, APG00356 (84.81% 99 identity, 89.11% similarity) APG00568(84.56% identity, 89.11% similarity) APG00287 (83.8% identity, 88.1%similarity) APG00157 (83.04% identity, 88.1% similarity) APG00377(82.78% identity, 88.35% similarity) APG00231 (82.53% identity, 87.85%similarity) WP_050845516.1 (80.76% identity, 86.84% similarity)AEX56523.1 (80.51% identity, 86.33% similarity) APG00494 (75.19%identity, 82.78% similarity) WP_000143307.1 (75.19% identity, 83.29%similarity) Cry35Ac2 (21.33% identity, 37.61% similarity) APG00781 146147 Cry68 75, 80, 85, 90, 95, 96, 97, 85, 90, 95, 96, 97, WP_016083794.198, 99 98, 99 (71.85% identity, 82.25% similarity) APG00026 (71.2%identity, 80.64% similarity) CA_2753918-14 (70.55% identity, 79.88%similarity) APG00109 (68.01% identity, 77.56% similarity) AFU17323.1(66.24% identity, 74.1% similarity) Cry68Aa1 (66.13% identity, 73.99%similarity) APG00784 148 149 Cry 35, 40, 45, 50, 55, 60, 65, 50, 55, 60,65, 70, APG00099 (95.29% 70, 75, 80, 85, 90, 95, 96, 75, 80, 85, 90, 95,identity, 96.86% 97, 98, 99 96, 97, 98, 99 similarity) APG00801 (84.55%identity, 90.37% similarity) Cry13Aa1 (30.61% identity, 45.66%similarity) APG00785 150 151, 152 Cry50 80, 85, 90, 95, 96, 97, 98, 90,95, 96, 97, 98, AGA40023.1 (77.24% 99 99 identity, 85.06% similarity)US20130227743A1_78 (61.79% identity, 74.2% similarity) KIQ78153.1(61.54% identity, 73.57% similarity) Cry50Ba1 (50.9% identity, 63.25%similarity) APG00786 153 154, 155 Cry69 95, 96, 97, 98, 99 95, 96, 97,98, 99 US_2011_0197314_A1- 13 (91.13% identity, 92.98% similarity)CA_2753918-13 (90.92% identity, 92.77% similarity) APG00662 (70.46%identity, 77.45% similarity) APG00079 (68.76% identity, 76.11%similarity) WP_016084057.1 (64.86% identity, 74.7% similarity) APG00059(64.27% identity, 74.75% similarity) Cry69Aa1 (59.64% identity, 69.36%similarity) APG00787 156 157 Cry 40, 45, 50, 55, 60, 65, 70, 55, 60, 65,70, 75, APG00723 (50.82% 75, 80, 85, 90, 95, 96, 97, 80, 85, 90, 95, 96,identity, 62.48% 98, 99 97, 98, 99 similarity) WP_048536362.1 (38.59%identity, 52.5% similarity) AGA40057.1 (33.33% identity, 46.48%similarity) WP_017762581.1 (29.66% identity, 40.4% similarity)AGA40058.1 (28.99% identity, 42.17% similarity) APG00799 158 159 Cry 25,30, 35, 40, 45, 50, 55, 40, 45, 50, 55, 60, C0ZKJ5_BREBN 60, 65, 70, 75,80, 85, 90, 65, 70, 75, 80, 85, (23.19% identity, 95, 96, 97, 98, 99 90,95, 96, 97, 98, 37.57% similarity) 99 Cry5Ad1 (21.24% identity, 32.24%similarity) APG00801 160 161 Cry 30, 35, 40, 45, 50, 55, 60, 50, 55, 60,65, 70, APG00099 (85.30% 65, 70, 75, 80, 85, 90, 95, 75, 80, 85, 90, 95,identity, 90.68% 96, 97, 98, 99 96, 97, 98, 99 similarity) APG00784(84.55% identity, 90.37% similarity) Cry13Aa1 (29.5% identity, 45.1%similarity) APG00802 162 163 Cry19 55, 60, 65, 70, 75, 80, 85, 70, 75,80, 85, 90, Cry19Ba1 (51.48% 90, 95, 96, 97, 98, 99 95, 96, 97, 98, 99identity, 67.28% similarity) AGV55021.1 (48.09% identity, 61.10%similarity) Cry52Aa1 (42.10% identity, 57.08% similarity) Cry19Aa1(40.17% identity, 55.23% similarity) ACP43735.1 (40.03% identity, 54.79%similarity) APG00805 164 165, 166 Cry40 60, 65, 70, 75, 80, 85, 90, 70,75, 80, 85, 90, WP_050845421.1 95, 96, 97, 98, 99 95, 96, 97, 98, 99(56.11% identity, 69.03% similarity) WO_2015_039599-9 (53.89% identity,66.25% similarity) Cry40Ba1 (48.4% identity, 60.89% similarity) APG00806167 168 Bin 85, 90, 95, 96, 97, 98, 99 95, 96, 97, 98, 99US20130227743A1_146 (84.73% identity, 90.39% similarity) APG00212(84.71% identity, 88.59% similarity) APG00592 (80.34% identity, 85.92%similarity) APG00619 (80.2% identity, 87.38% similarity) APG00600(79.65% identity, 87.1% similarity) APG00798 (79.42% identity, 85.47%similarity) WP_002166959.1 (74.0% identity, 83.0% similarity)WP_002191947.1 (73.75% identity, 83.0% similarity) APG00724 (67.08%identity, 79.7% similarity) APG00716 (66.83% identity, 76.66%similarity) APG00701 (64.75% identity, 77.75% similarity) APG00988(59.25% identity, 71.25% similarity) Cry35Ac2 (21.85% identity, 35.32%similarity) APG00807 169 170 Mtx 35, 40, 45, 50, 55, 60, 65, 50, 55, 60,65, 70, APG00641 (73.68% 70, 75, 80, 85, 90, 95, 96, 75, 80, 85, 90, 95,identity, 82.57% 97, 98, 99 96, 97, 98, 99 similarity) APG00434 (52.88%identity, 68.59% similarity) Cry64Aa1 (32.69% identity, 48.08%similarity) APG00810 171 172, 173 Cry20 60, 65, 70, 75, 80, 85, 90, 70,75, 80, 85, 90, AGV55017.1 (57.12% 95, 96, 97, 98, 99 95, 96, 97, 98, 99identity, 68.39% similarity) WP_016098327.1 (53.01% identity, 64.12%similarity) Cry20Ba2 (52.47% identity, 62.58% similarity) APG00864 174Cry54A 85, 90, 95, 96, 97, 98, 99 95, 96, 97, 98, 99 AGA40050.1 (84.65%identity, 90.2% similarity) Cry54Aa2 (83.63% identity, 89.18%similarity) APG00664 (58.9% identity, 70.48% similarity) APG00912 175176, 177 178 Cry 70, 75, 80, 85, 90, 95, 96, 80, 85, 90, 95, 96,AEH76820.1 (69.56% 97, 98, 99 97, 98, 99 identity, 78.62% similarity)US20100298211A1_8 (65.27% identity, 73.73% similarity)US20130227743A1_48 (55.33% identity, 70.45% similarity) Cry32Ea1 (44.82%identity, 57.88% similarity) APG00960 179 180, 181 Cry7 95, 96, 97, 98,99 95, 96, 97, 98, 99 AGU13819.1 (90.49% identity, 94.42% similarity)AGM39662.1 (88.66% identity, 93.37% similarity) AGU13834.1 (87.78%identity, 93.28% similarity) Cry7Ab3 (58.23% identity, 69.62%similarity) APG00972 182 183, 184 Cry30F 90, 95, 96, 97, 98, 99 95, 96,97, 98, 99 CA_2753918-15 (85.29% identity, 90.76% similarity) AFU17333.1(84.87% identity, 90.48% similarity) WP_000806152.1 (81.65% identity,87.25% similarity) Cry30Fa1 (80.81% identity, 86.69% similarity)APG00565 (63.5% identity, 75.94% similarity) APG00980 185 186, 187 Mtx65, 70, 75, 80, 85, 90, 95, 75, 80, 85, 90, 95, AGA40045.1 (61.81% 96,97, 98, 99 96, 97, 98, 99 identity, 71.14% similarity) APG00351 (58.84%identity, 72.46% similarity) APG00939 (58.52% identity, 69.32%similarity) US20130227743A1_102 (56.65% identity, 69.08% similarity)APG00146 (55.46% identity, 69.32% similarity) APG00387 (55.3% identity,67.91% similarity) WP_000794514.1 (54.28% identity, 69.32% similarity)APG00938 (51.83% identity, 65.07% similarity) WP_036654376.1 (41.38%identity, 56.32% similarity) APG00981 188 189 Cry 40, 45, 50, 55, 60,65, 70, 55, 60, 65, 70, 75, APG00076 (85.87% 75, 80, 85, 90, 95, 96, 97,80, 85, 90, 95, 96, identity, 91.14% 98, 99 97, 98, 99 similarity)CA_2843744-7 (39.07% identity, 54.08% similarity) CA_2843744-9 (38.92%identity, 54.29% similarity) US20130227743A1_74 (34.23% identity, 50.19%similarity) Cry4Cc1 (21.66% identity, 32.04% similarity) APG00986 190191, 192 193 Cry56 85, 90, 95, 96, 97, 98, 99 90, 95, 96, 97, 98,AGV55019.1 (81.41% 99 identity, 86.81% similarity) ACR88315.1 (63.53%identity, 75.44% similarity) WP_050845711.1 (54.32% identity, 69.79%similarity) Cry56Aa2 (54.3% identity, 69.73% similarity) APG00988 194195 Bin 75, 80, 85, 90, 95, 96, 97, 90, 95, 96, 97, 98, WP_002114997.198, 99 99 (73.67% identity, 81.01% similarity) WP_002187944.1 (73.67%identity, 85.06% similarity) WP_001258160.1 (72.91% identity, 84.56%similarity) APG00213 (72.49% identity, 82.26% similarity) APG00243(72.35% identity, 82.43% similarity) APG00412 (71.21% identity, 80.56%similarity) APG00844 (70.28% identity, 81.65% similarity) APG00118(67.96% identity, 76.43% similarity) APG00648 (67.83% identity, 77.06%similarity) APG00716 (65.9% identity, 76.41% similarity) APG00724(60.78% identity, 71.32% similarity) APG00806 (59.25% identity, 71.25%similarity) Cry35Ab4 (23.15% identity, 40.33% similarity) APG01000 196197 Bin 30, 35, 40, 45, 50, 55, 60, 45, 50, 55, 60, 65, WP_002090518.165, 70, 75, 80, 85, 90, 95, 70, 75, 80, 85, 90, (26.42% identity, 96,97, 98, 99 95, 96, 97, 98, 99 40.16% similarity) WP_048517129.1 (26.3%identity, 40.55% similarity) WP_016093722.1 (26.22% identity, 41.35%similarity) Cry49Ab1 (16.28% identity, 24.74% similarity) APG01003 198199 Vip3 80, 85, 90, 95, 96, 97, 98, 90, 95, 96, 97, 98, APG00875(87.19% 99 99 identity, 90.21% similarity) APG00278 (85.25% identity,90.06% similarity) APG00173 (85.18% identity, 89.77% similarity)APG00358 (81.35% identity, 87.4% similarity) APG00273 (78.16% identity,85.2% similarity) WP_050001316.1 (75.08% identity, 85.24% similarity)APG00939 (72.64% identity, 80.23% similarity) WP_048517127.1 (72.11%identity, 79.78% similarity) APG00657 (67.97% identity, 78.06%similarity) AIT93175.1 (23.47% identity, 39.55% similarity) Vip3Ad2(23.32% identity, 39.53% similarity) APG01028 200 201, 202 203 Cry 85,90, 95, 96, 97, 98, 99 90, 95, 96, 97, 98, CA_2753918-17 99 (81.0%identity, 87.71% similarity) APG00555 (66.91% identity, 76.83%similarity) US20130227743A1_26 (61.01% identity, 72.81% similarity)APG00606 (54.05% identity, 66.71% similarity) ACP43734.1 (41.05%identity, 55.06% similarity) Cry53Aa1 (40.14% identity, 54.69%similarity) APG01112 204 Cry 30, 35, 40, 45, 50, 55, 60, 45, 50, 55, 60,65, APG00603 (79.84% 65, 70, 75, 80, 85, 90, 95, 70, 75, 80, 85, 90,identity, 81.48% 96, 97, 98, 99 95, 96, 97, 98, 99 similarity)WP_017762616.1 (26.86% identity, 41.34% similarity) WP_044306756.1(26.77% identity, 40.34% similarity) US20130227743A1_206 (23.74%identity, 37.77% similarity) AGA40058.1 (22.9% identity, 35.14%similarity) APG00556 205 206 Mtx 65, 70, 75, 80, 85, 90, 95, 80, 85, 90,95, 96, APG00623 (88.97% 96, 97, 98, 99 97, 98, 99 identity, 91.18%similarity) APG01037 (88.97% identity, 91.54% similarity)US_2014_0283208_A1- 2 (61.29% identity, 76.34% similarity)WO_2014_159836-52 (60.93% identity, 76.34% similarity) WO_2014_159836-61(60.93% identity, 76.34% similarity) Cry46Ab (38.31% identity, 56.17%similarity) APG00623 207 Mtx 65, 70, 75, 80, 85, 90, 95, 85, 90, 95, 96,97, APG01037 (98.02% 96, 97, 98, 99 98, 99 identity, 99.21% similarity)APG00556 (88.97% identity, 91.18% similarity) US_2014_0283208_A1- 2(64.34% identity, 81.4% similarity) WO_2014_159836-52 (63.95% identity,81.4% similarity) WO_2014_159836-61 (63.95% identity, 81.4% similarity)Cry46Ab (35.83% identity, 52.77% similarity) APG01037 208 209, 210, Mtx65, 70, 75, 80, 85, 90, 95, 85, 90, 95, 96, 97, APG00623 (98.02% 211,212, 96, 97, 98, 99 98, 99 identity, 99.21% 213, 214 similarity)APG00556 (88.97% identity, 91.54% similarity) US_2014_0283208_A1- 2(64.48% identity, 81.08% similarity) WO_2014_159836-52 (64.09% identity,81.08% similarity) WO_2014_159836-61 (64.09% identity, 81.08%similarity) Cry46Ab (36.51% identity, 53.29% similarity) APG01086 215216 Mtx 75, 80, 85, 90, 95, 96, 97, 85, 90, 95, 96, 97, APG00566 (97.39%98, 99 98, 99 identity, 99.02% similarity) APG00201 (81.05% identity,90.85% similarity) APG00006 (78.76% identity, 90.20% similarity)APG00260 (78.10% identity, 87.58% similarity) APG00036 (75.24% identity,83.71% similarity) APG00022 (75.16% identity, 83.01% similarity)WP_000963933.1 (74.18% identity, 84.31% similarity) US_2013_0227743_A1-100 (72.88% identity, 83.66% similarity) US_2013_0227743_A1- 99 (72.64%identity, 83.39% similarity) APG00345 (68.08% identity, 80.46%similarity) APG06508 217 Mtx 85, 90, 95, 96, 97, 98, 99 95, 96, 97, 98,99 APG00661 (96.41% identity, 97.31% similarity) APG09801 (95.47%identity, 96.68% similarity) US_8829279_B2-24 (84.13% identity, 90.12%similarity) US_8829279_B2-34 (75.60% identity, 84.94% similarity)US_8318900_B2-13 (75.30% identity, 84.64% similarity) APG09801 218 Mtx85, 90, 95, 96, 97, 98, 99 90, 95, 96, 97, 98, APG06508 (95.47% 99identity, 96.68% similarity) APG00661 (94.91% identity, 97.01%similarity) US_8829279_B2-24 (81.74% identity, 89.22% similarity)US_8829279_B2-34 (76.28% identity, 84.98% similarity) US_8318900_B2-13(75.98% identity, 84.68% similarity) APG01037 208 60, 61, 62, 63, 64,65, 60, 61, 62, 63, 66, 67, 68, 69, 70, 71, 64, 65, 66, 67, 72, 73, 74,75, 76, 77, 68, 69, 70, 75, 78, 79, 80, 81, 82, 83, 80, 85, 86, 87, 84,85, 86, 87, 88, 89, 88, 89, 90, 91, 90, 91, 92, 93, 94, 95, 92, 93, 94,96, 97, 98, 99 95, 96, 97, 98, 99 APG01037.1 209 60, 61, 62, 63, 64, 65,60, 61, 62, 63, 66, 67, 68, 69, 70, 71, 64, 65, 66, 67, 72, 73, 74, 75,76, 77, 68, 69, 70, 75, 78, 79, 80, 81, 82, 83, 80, 85, 86, 87, 84, 85,86, 87, 88, 89, 88, 89, 90, 91, 90, 91, 92, 93, 94, 95, 92, 93, 94, 95,96, 97, 98, 99 96, 97, 98, 99 APG00623 207 60, 61, 62, 63, 64, 65, 60,61, 62, 63, 66, 67, 68, 69, 70, 71, 64, 65, 66, 67, 72, 73, 74, 75, 76,77, 68, 69, 70, 75, 78, 79, 80, 81, 82, 83, 80, 85, 86, 87, 84, 85, 86,87, 88, 89, 88, 89, 90, 91, 90, 91, 92, 93, 94, 95, 92, 93, 94, 95, 96,97, 98, 99 96, 97, 98, 99 APG00556 205 60, 61, 62, 63, 64, 65, 60, 61,62, 63, 66, 67, 68, 69, 70, 71, 64, 65, 66, 67, 72, 73, 74, 75, 76, 77,68, 69, 70, 75, 78, 79, 80, 81, 82, 83, 80, 85, 86, 87, 84, 85, 86, 87,88, 89, 88, 89, 90, 91, 90, 91, 92, 93, 94, 95, 92, 93, 94, 96, 97, 98,99 95, 96, 97, 98, 99 APG00556.1 206 60, 61, 62, 63, 64, 65, 60, 61, 62,63, 66, 67, 68, 69, 70, 71, 64, 65, 66, 67, 72, 73, 74, 75, 76, 77, 68,69, 70, 75, 78, 79, 80, 81, 82, 83, 80, 85, 86, 87, 84, 85, 86, 87, 88,89, 88, 89, 90, 91, 90, 91, 92, 93, 94, 95, 92, 93, 94, 95, 96, 97, 98,99 96, 97, 98, 99

i. Classes of Pesticidal Proteins

The pesticidal proteins provided herein and the nucleotide sequencesencoding them are useful in methods for impacting pests. That is, thecompositions and methods of the invention find use in agriculture forcontrolling or killing pests, including pests of many crop plants. Thepesticidal proteins provided herein are toxin proteins from bacteria andexhibit activity against certain pests. The pesticidal proteins are fromseveral classes of toxins including Cry, Cyt, BIN, Mtx toxins. See, forexample, Table 1 for the specific protein classifications of the variousSEQ ID NOS provided herein. In addition, reference is made throughoutthis disclosure to Pfam database entries. The Pfam database is adatabase of protein families, each represented by multiple sequencealignments and a profile hidden Markov model. Finn et al. (2014) Nucl.Acid Res. Database Issue 42:D222-D230.

Bacillus thuringiensis (Bt) is a gram-positive bacterium that producesinsecticidal proteins as crystal inclusions during its sporulation phaseof growth. The proteinaceous inclusions of Bacillus thuringiensis (Bt)are called crystal proteins or δ-endotoxins (or Cry proteins), which aretoxic to members of the class Insecta and other invertebrates.Similarly, Cyt proteins are parasporal inclusion proteins from Bt thatexhibits hemolytic (Cytolitic) activity or has obvious sequencesimilarity to a known Cyt protein. These toxins are highly specific totheir target organism, are innocuous to humans, vertebrates, and plants.

The structure of the Cry toxins reveals five conserved amino acidblocks, concentrated mainly in the center of the domain or at thejunction between the domains. The Cry toxin consists of three domains,each with a specific function. Domain I is a seven α-helix bundle inwhich a central helix is completely surrounded by six outer helices.This domain is implicated in channel formation in the membrane. DomainII appears as a triangular column of three anti-parallel β-sheets, whichare similar to antigen-binding regions of immunoglobulins. Domain IIIcontains anti-parallel β-strands in a β sandwich form. The N-terminalpart of the toxin protein is responsible for its toxicity andspecificity and contains five conserved regions. The C-terminal part isusually highly conserved and probably responsible for crystal formation.See, for example, U.S. Pat. No. 8,878,007.

Strains of B. thuringiensis show a wide range of specificity againstdifferent insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera,Homoptera, Phthiraptera or Mallophaga, and Acari) and otherinvertebrates (Nemathelminthes, Platyhelminthes, and Sarocomastebrates).The cry proteins have been classified into groups based on toxicity tovarious insect and invertebrate groups. Generally, Cry I demonstratestoxicity to lepidopterans, Cry II to lepidopterans and dipterans, CryIIIto coleopterans, Cry IV to dipterans, and Cry V and Cry VI to nematodes.New Cry proteins can be identified and assigned to a Cry group based onamino acid identity. See, for example, Bravo, A. (1997) J. of Bacteria179:2793-2801; Bravo et al. (2013) Microb. Biotechnol. 6:17-26, hereinincorporated by reference.

Over 750 different cry gene sequences have been classified into 73groups (Cry1-Cry73), with new members of this gene family continuing tobe discovered (Crickmore et al. (2014) www.btnomenclature.info/). Thecry gene family consists of several phylogentically non-related proteinfamilies that may have different modes of action: the family ofthree-domain Cry toxins, the family of mosquitocidal Cry toxins, thefamily of the binary-like toxins, and the Cyt family of toxins (Bravo etal., 2005). Some Bt strains produce additional insecticidal toxins, theVIP toxins. See, also, Cohen et al. (2011) J. Mol. Biol. 413:4-814;Crickmore et al. (2014) Bacillus thuringiensis toxin nomenclature, foundon the world wide web at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/;Crickmore et al. (1988) Microbiol. Mol. Biol. Rev. 62: 807-813; Gill etal. (1992) Ann. Rev. Entomol. 37: 807-636; Goldbert et al. (1997) Appl.Environ. Microbiol. 63:2716-2712; Knowles et al. (1992) Proc. R. Soc.Ser. B. 248: 1-7; Koni et al. (1994) Microbiology 140: 1869-1880; Lailaket al. (2013) Biochem. Biophys. Res. Commun. 435: 216-221; Lopez-Diaz etal. (2013) Environ. Microbiol. 15: 3030-3039; Perez et al. (2007) Cell.Microbiol. 9: 2931-2937; Promdonkoy et al. (2003) Biochem. J. 374:255-259; Rigden (2009) FEBS Lett. 583: 1555-1560; Schnepf et al. (1998)Microbiol. Mol. Biol. Rev. 62: 775-806; Soberon et al. (2013) Peptides41: 87-93; Thiery et al. (1998) J. Am. Mosq. Control Assoc. 14: 472-476;Thomas et al. (1983) FEBS Lett. 154: 362-368; Wirth et al. (1997) Proc.Natl. Acad. Sci. U.S.A. 94: 10536-10540; Wirth et al (2005) Appl.Environ. Microbiol. 71: 185-189; and, Zhang et al. (2006) Biosci.Biotechnol. Biochem. 70: 2199-2204; each of which is herein incorporatedby reference in their entirety.

Cyt designates a parasporal crystal inclusion protein from Bacillusthuringiensis with cytolytic activity, or a protein with sequencesimilarity to a known Cyt protein. (Crickmore et al. (1998) Microbiol.Mol. Biol. Rev. 62: 807-813). The gene is denoted by cyt. These proteinsare different in structure and activity from Cry proteins (Gill et al.(1992) Annu. Rev. Entomol. 37: 615-636). The Cyt toxins were firstdiscovered in B. thuringiensis subspecies israelensis (Goldberg et al.(1977) Mosq. News. 37: 355-358). There are 3 Cyt toxin familiesincluding 11 holotype toxins in the current nomenclature (Crickmore etal. (2014) Bacillus thuringiensis toxin nomenclature found on the worldwide web at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). The majorityof the B. thuringiensis isolates with cyt genes show activity againstdipteran insects (particularly mosquitoes and black flies), but thereare also cyt genes that have been described in B. thuringiensis strainstargeting lepidopteran or coleopteran insects (Guerchicoff et al. (1997)Appl. Environ. Microbiol. 63: 2716-2721).

The structure of Cyt2A, solved by X-ray crystallography, shows a singledomain where two outer layers of α-helix wrap around a mixed β-sheet.Further available crystal structures of Cyt toxins support a conservedα-β structural model with two α-helix hairpins flanking a β-sheet corecontaining seven to eight β-strands. (Cohen et al. (2011) J. Mol. Biol.413: 80 4-814) Mutagenic studies identified β-sheet residues as criticalfor toxicity, while mutations in the helical domains did not affecttoxicity (Adang et al.; Diversity of Bacillus thuringiensis CrystalToxins and Mechanism of Action. In: T. S. Dhadialla and S. S. Gill, eds,Advances in Insect Physiology, Vol. 47, Oxford: Academic Press, 2014,pp. 39-87.) The representative domain of the Cyt toxin is a δ-endotoxin,Bac_thur_toxin (Pfam PF01338).

There are multiple proposed models for the mode of action of Cyt toxins,and it is still an area of active investigation. Some Cyt proteins(Cyt1A) have been shown to require the presence of accessory proteinsfor crystallization. Cyt1A and Cyt2A protoxins are processed bydigestive proteases at the same sites in the N- and C-termini to astable toxin core. Cyt toxins then interact with non-saturated membranelipids, such as phosphatidylcholine, phosphatidylethanolamine, andsphingomyelin. For Cyt toxins, pore-formation and detergent-likemembrane disruption have been proposed as non-exclusive mechanisms; andit is generally accepted that both may occur depending on toxinconcentration, with lower concentrations favoring oligomeric pores andhigher concentrations leading to membrane breaks. (Butko (2003) Appl.Environ. Microbiol. 69: 2415-2422) In the pore-formation model, the Cyttoxin binds to the cell membrane, inducing the formation ofcation-selective channels in the membrane vesicles leading tocolloid-osmotic lysis of the cell. (Knowles et al. (1989) FEBS Lett.244: 259-262; Knowles et al. (1992) Proc. R. Soc. Ser. B. 248: 1-7 andPromdonkoy et al. (2003) Biochem. J. 374: 255-259). In the detergentmodel, there is a nonspecific aggregation of the toxin on the surface ofthe lipid bilayer leading to membrane disassembly and cell death. (Butko(2003) supra; Manceva et al. (2005) Biochem. 44: 589-597).

Multiple studies have shown synergistic activity between Cyt toxins andother B. thuringiensis toxins, particularly the Cry, Bin, and Mtxtoxins. This synergism has even been shown to overcome an insect'sresistance to the other toxin. (Wirth 1997, Wirth 2005, Thiery 1998,Zhang 2006) The Cyt synergistic effect for Cry toxins is proposed toinvolve Cyt1A binding to domain II of Cry toxins in solution or on themembrane plane to promote formation of a Cry toxin pre-pore oligomer.Formation of this oligomer is independent of the Cyt oligomerization,binding or insertion. (Lailak 2013, Perez 2007, Lopez-Diaz 2013)

A number of pesticidal proteins unrelated to the Cry proteins areproduced by some strains of B. thuringiensis and B. cereus duringvegetative growth (Estruch et al. (1996) Proc Natl Acad Sci USA93:5389-5394; Warren et al. (1994) WO 94/21795). These vegetativeinsecticidal proteins, or Vips, do not form parasporal crystal proteinsand are apparently secreted from the cell. The Vips are presentlyexcluded from the Cry protein nomenclature because they are notcrystal-forming proteins. The term VIP is a misnomer in the sense thatsome B. thuringiensis Cry proteins are also produced during vegetativegrowth as well as during the stationary and sporulation phases, mostnotably Cry3Aa. The location of the Vip genes in the B. thuringiensisgenome has been reported to reside on large plasmids that also encodecry genes (Mesrati et al. (2005) FEMS Microbiol. Lett. 244(2):353-8). Aweb-site for the nomenclature of Bt toxins can be found on the worldwide web at lifesci.sussex.ac.uk with the path“/home/Neil_Crickmore/Bt/” and at: “btnomenclature.info/”. See also,Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62(3):775-806. Suchreferences are herein incorporated by reference.

To date four categories of Vips have been identified. Some Vip genesform binary two-component protein complexes; an “A” component is usuallythe “active” portion, and a “B” component is usually the “binding”portion. (Pfam pfam.xfam.org/family/PF03495). The Vip1 and Vip4 proteinsgenerally contain binary toxin B protein domains. Vip2 proteinsgenerally contain binary toxin A protein domains.

The Vip1 and Vip2 proteins are the two components of a binary toxin thatexhibits toxicity to coleopterans. Vip1Aa1 and Vip2Aa1 are very activeagainst corn rootworms, particularly Diabrotica virgifera and Diabroticalongicornis (Han et al. (1999) Nat. Struct. Biol. 6:932-936; Warren G W(1997) “Vegetative insecticidal proteins: novel proteins for control ofcorn pests” In: Carozzi N B, Koziel M (eds) Advances in insect control,the role of transgenic plants; Taylor & Francis Ltd, London, pp 109-21).The membrane-binding 95 kDa Vip1 multimer provides a pathway for the 52kDa vip2 ADP-ribosylase to enter the cytoplasm of target western cornrootworm cells (Warren (1997) supra). The NAD-dependentADP-ribosyltransferase Vip2 likely modifies monomeric actin at Arg177 toblock polymerization, leading to loss of the actin cytoskeleton andeventual cell death due to the rapid subunit ex-change within actinfilaments in vivo (Carlier M. F. (1990) Adv. Biophys. 26:51-73).

Like Cry toxins, activated Vip3A toxins are pore-forming proteinscapable of making stable ion channels in the membrane (Lee et al. (2003)Appl. Environ. Microbiol. 69:4648-4657). Vip3 proteins are activeagainst several major lepidopteran pests (Rang et al. (2005) Appl.Environ. Microbiol. 71(10):6276-6281; Bhalla et al. (2005) FEMSMicrobiol. Lett. 243:467-472; Estruch et al. (1998) WO 9844137; Estruchet al. (1996) Proc Natl Acad Sci USA 93:5389-5394; Selvapandiyan et al.(2001) Appl. Environ Microbiol. 67:5855-5858; Yu et al. (1997) Appl.Environ Microbiol. 63:532-536). Vip3A is active against Agrotis ipsilon,Spodoptera frugiperda, Spodoptera exigua, Heliothis virescens, andHelicoverpa zea (Warren et al. (1996) WO 96/10083; Estruch et al. (1996)Proc Natl Acad Sci USA 93:5389-5394). Like Cry toxins, Vip3A proteinsmust be activated by proteases prior to recognition at the surface ofthe midgut epithelium of specific membrane proteins different from thoserecognized by Cry toxins.

The MTX family of toxin proteins is characterized by the presence of aconserved domain, ETX_MTX2 (pfam 03318). Members of this family sharesequence homology with the mosquitocidal toxins Mtx2 and Mtx3 fromBacillus sphaericus, as well as with the epsilon toxin ETX fromClostridium perfringens (Cole et al. (2004) Nat. Struct. Mol. Biol. 11:797-8; Thanabalu et al. (1996) Gene 170:85-9). The MTX-like proteins arestructurally distinct from the three-domain Cry toxins, as they have anelongated and predominately β-sheet-based structure. However, similar tothe three-domain toxins, the MTX-like proteins are thought to form poresin the membranes of target cells (Adang et al. (2014) supra). Unlike thethree-domain Cry proteins, the MTX-like proteins are much smaller inlength, ranging from 267 amino acids (Cry23) to 340 amino acids (Cry15A.

To date, only 15 proteins belonging to the family of MTX-like toxinshave been assigned Cry names, making this a relatively small classcompared to the three-domain Cry family (Crickmore et al. (2014) supra;Adang et al. (2014) supra). The members of the MTX-like toxin familyinclude Cry15, Cry23, Cry33, Cry38, Cry45, Cry46, Cry51, Cry60A, Cry60B,and Cry64. This family exhibits a range of insecticidal activity,including activity against insect pests of the Lepidopteran andColeopteran orders. Some members of this family may form binarypartnerships with other proteins, which may or may not be required forinsecticidal activity.

Cry15 is a 34 kDA protein that was identified in Bacillus thuringiensisserovar thompsoni HD542; it occurs naturally in a crystal together withan unrelated protein of approximately 40 kDa. The gene encoding Cry15and its partner protein are arranged together in an operon. Cry15 alonehas been shown to have activity against lepidopteran insect pestsincluding Manduca sexta, Cydia pomonella, and Pieris rapae, with thepresence of the 40 kDA protein having been shown to increase activity ofCry15 only against C. pomonella (Brown K. and Whiteley H. (1992) J.Bacteriol. 174:549-557; Naimov et al. (2008) Appl. Environ. Microbiol.74:7145-7151). Further studies are needed to elucidate the function ofthe partner protein of Cry15. Similarly, Cry23 is a 29 kDA protein thathas been shown to have activity against the coleopteran pests Triboliumcastaneum and Popillia japonica together with its partner protein Cry37(Donovan et al. (2000) U.S. Pat. No. 6,063,756).

New members of the MTX-like family are continuing to be identified. AnETX_MTX toxin gene was recently identified in the genome of Bacillusthuringiensis serovar tolworthi strain Na205-3. This strain was found tobe toxic against the lepidpoteran pest Helicoverpa armigera, and it alsocontained homologs of Cry1, Cry11, Vip1, Vip2, and Vip3 (Palma et al.(2014) Genome Announc. 2(2): e00187-14. Published online Mar. 13, 2014at doi: 10.1128/genomeA.00187-14; PMCID: PMC3953196). Because theMTX-like proteins have a unique domain structure relative to thethree-domain Cry proteins, they are believed to possess a unique mode ofaction, thereby making them a valuable tool in insect control and thefight against insect resistance. Bacterial cells produce large numbersof toxins with diverse specificity against host and non-host organisms.Large families of binary toxins have been identified in numerousbacterial families, including toxins that have activity against insectpests. (Poopathi and Abidha (2010) J. Physiol. Path. 1(3): 22-38).Lysinibacillus sphaericus (Ls), formerly Bacillus sphaericus, (Ahmed etal. (2007) Int. J. Syst. Evol. Microbiol. 57:1117-1125) is well-known asan insect biocontrol strain. Ls produces several insecticidal proteins,including the highly potent binary complex BinA/BinB. This binarycomplex forms a parasporal crystal in Ls cells and has strong andspecific activity against dipteran insects, specifically mosquitoes. Insome areas, insect resistance to existing Ls mosquitocidal strains hasbeen reported. The discovery of new binary toxins with different targetspecificity or the ability to overcome insect resistance is ofsignificant interest.

The Ls binary insecticidal protein complex contains two majorpolypeptides, a 42 kDa polypeptide and a 51 kDa polypeptide, designatedBinA and BinB, respectively (Ahmed et al. (2007) supra). The twopolypeptides act synergistically to confer toxicity to their targets.Mode of action involves binding of the proteins to receptors in thelarval midgut. In some cases, the proteins are modified by proteasedigestion in the larval gut to produce activated forms. The BinBcomponent is thought to be involved in binding, while the BinA componentconfers toxicity (Nielsen-LeRoux et al. (2001) Appl. Environ. Microbiol.67(11):5049-5054). When cloned and expressed separately, the BinAcomponent is toxic to mosquito larvae, while the BinB component is not.However, co-administration of the proteins markedly increases toxicity(Nielsen-LeRoux et al. (2001) supra).

A small number of Bin protein homologs have been described frombacterial sources. Priest et al. (1997) Appl. Environ. Microbiol.63(4):1195-1198 describe a hybridization effort to identify new Lsstrains, although most of the genes they identified encoded proteinsidentical to the known BinA/BinB proteins. The BinA protein contains adefined conserved domain known as the Toxin 10 superfamily domain. Thistoxin domain was originally defined by its presence in BinA and BinB.The two proteins both have the domain, although the sequence similaritybetween BinA and BinB is limited in this region (<40%). The Cry49Aaprotein, which also has insecticidal activity, also has this domain(described below).

The Cry48Aa/Cry49Aa binary toxin of Ls has the ability to kill Culexquinquefasciatus mosquito larvae. These proteins are in a proteinstructural class that has some similarity to the Cry protein complex ofBacillus thuringiensis (Bt), a well-known insecticidal protein family.The Cry34/Cry35 binary toxin of Bt is also known to kill insects,including Western corn rootworm, a significant pest of corn. Cry34, ofwhich several variants have been identified, is a small (14 kDa)polypeptide, while Cry35 (also encoded by several variants) is a 44 kDapolypeptide. These proteins have some sequence homology with theBinA/BinB protein group and are thought to be evolutionarily related(Ellis et al. (2002) Appl. Environ. Microbiol. 68(3):1137-1145).

Phosphoinositide phospholipase C proteins (PI-PLC; alsophosphotidylinositol phospholipase C) are members of the broader groupof phospholipase C proteins. Many of these proteins play important rolesin signal transduction as part of normal cell physiology. Severalimportant bacterial toxins also contain domains with similarity to theseproteins (Titball, R. W. (1993) Microbiological Reviews. 57(2):347-366).Importantly, these proteins are implicated in signal amplificationduring intoxication of insect cells by Bt Cry proteins (Valaitis, A. P.(2008) Insect Biochemistry and Molecular Biology. 38: 611-618).

The PI-PLC toxin class occurs in Bacillus isolates, commonly seen inco-occurrence with homologs to other described toxin classes, such asBinary Toxins. This class of sequences has homology tophosphatidylinositol phosphodiesterases (also referred to asphosphatidylinositol-specific phospholipase C—PI-PLC). The crystalstructure and its active site were solved for B. cereus PI-PLC by Heinzet al (Heinz, et. al., (1995) The EMBO Journal. 14(16): 3855-3863). Theroles of the B. cereus PI-PLC active site amino acid residues incatalysis and substrate binding were investigated by Gässler et al usingsite-directed mutagenesis, kinetics, and crystal structure analysis(Gässler, et. al., (1997) Biochemistry. 36(42):12802-13).

These PI-PLC toxin proteins contain a PLC-like phosphodiesterase, TIMbeta/alpha-barrel domain (IPR017946) and/or a Phospholipase C,phosphatidylinositol-specific, X domain (IPR000909) (also referred to asthe PI-PLC X-box domain). We have also seen proteins with these domainsin combination with other typical Bacillus protein toxin domains. Thislist includes most commonly a lectin domain (IPR000772), a sugar-bindingdomain that can be present in one or more copies and is thought to bindcell membranes, as well as the Insecticidal crystal toxin (IPR008872)(also referred to as Toxin10 or P42), which is the defining domain ofthe Binary Toxin.

Previously, toxins of this PI-PLC class were defined in U.S. Pat. No.8,318,900 B2 SEQ ID NOs 30 (DNA) and 79 (amino acid), in U.S. PatentPublication No. 20110263488A1 SEQ ID NOs 8 (DNA) and 9 (amino acid), andin U.S. Pat. No. 8,461,421B2 SEQ ID NOs 3 (DNA) and 63 (amino acid).

Provided herein are pesticidal proteins from these classes of toxins.The pesticidal proteins are classified by their structure, homology toknown toxins and/or their pesticidal specificity. Table 2 provides thePFAM domains present in some of the recited SEQ ID NOS.

Further provided are APG01037.1 is set forth in SEQ ID NO:209 and it isa fragment of SEQ ID NO: 208 (APG01037); shares 98% sequence identity toSEQ ID NO: 207 (APG00623); shares 96% sequence identity to SEQ ID NO:206 (APG00556.1); and shares 96% sequence identity to SEQ ID NO: 205(APG00556).

TABLE 2 PFAM domains Domain Modification PFAM Domain positions APG IDSeq ID Type domain Description Start Stop APG00326 Seq ID 1 PF03945Endotoxin N 87 332 PF00555 Endotoxin M 337 543 PF03944 Endotoxin C 553697 APG00326 Seq ID 2 Alternate start PF03945 Endotoxin N 70 315modified PF00555 Endotoxin M 320 526 PF03944 Endotoxin C 536 680APG00326 Seq ID 3 Alternate start PF03945 Endotoxin N 70 315 modifiedand 3′ PF00555 Endotoxin M 320 526 Truncation PF03944 Endotoxin C 536679 APG00343 Seq ID 4 PF03945 Endotoxin N 84 307 PF00555 Endotoxin M 312530 PF03944 Endotoxin C 540 677 APG00343 Seq ID 5 Alternate startPF03945 Endotoxin N 72 295 modified PF00555 Endotoxin M 300 518 PF03944Endotoxin C 528 665 APG00383 Seq ID 6 PF03945 Endotoxin N 78 329 PF03944Endotoxin C 557 717 APG00383 Seq ID 7 3′ Truncation PF03945 Endotoxin N78 329 modified PF03944 Endotoxin C 557 716 APG00493 Seq ID 8 no PFAMdomains APG00493 Seq ID 9 Alternate start no PFAM modified domainsAPG00494 Seq ID 10 PF14200 Ricin B 48 150 Lectin 2 PF05431 Toxin 10 156353 APG00494 Seq ID 11 Alternate start PF14200 Ricin B 44 145 modifiedLectin 2 PF05431 Toxin 10 152 349 APG00495 Seq ID 12 PF03318 ETX MTX2 50310 APG00495 Seq ID 13 Signal peptide PF03318 ETX MTX2 8 267 modifiedremoved APG00513 Seq ID 14 PF03318 ETX MTX2 80 326 APG00514 Seq ID 15PF03945 Endotoxin N 84 307 PF00555 Endotoxin M 312 530 PF03944 EndotoxinC 540 677 APG00514 Seq ID 16 Alternate start PF03945 Endotoxin N 72 295modified PF00555 Endotoxin M 300 518 PF03944 Endotoxin C 528 665APG00524 Seq ID 17 PF03945 Endotoxin N 66 316 PF00555 Endotoxin M 321523 PF03944 Endotoxin C 541 681 APG00524 Seq ID 18 Alternate startPF03945 Endotoxin N 66 316 modified and 3′ PF00555 Endotoxin M 321 523Truncation PF03944 Endotoxin C 541 680 APG00524 Seq ID 19 Alternatestart PF03945 Endotoxin N 66 316 modified PF00555 Endotoxin M 321 523PF03944 Endotoxin C 541 681 APG00528 Seq ID 20 no PFAM domains APG00533Seq ID 21 PF03945 Endotoxin N 37 232 APG00534 Seq ID 22 PF03945Endotoxin N 53 259 PF03945 Endotoxin N 290 331 PF00555 Endotoxin M 338544 PF03944 Endotoxin C 554 693 APG00534 Seq ID 23 3′ Truncation PF03945Endotoxin N 53 259 modified PF03945 Endotoxin N 290 331 PF00555Endotoxin M 338 544 PF03944 Endotoxin C 554 692 APG00536 Seq ID 24PF03945 Endotoxin N 103 336 PF00555 Endotoxin M 341 552 PF03944Endotoxin C 562 691 PF14200 Ricin B 731 833 Lectin 2 APG00536 Seq ID 25Alternate start PF03945 Endotoxin N 56 289 modified and 3′ PF00555Endotoxin M 294 505 Truncation PF03944 Endotoxin C 515 644 APG00536 SeqID 26 Alternate start PF03945 Endotoxin N 56 289 modified PF00555Endotoxin M 294 505 PF03944 Endotoxin C 515 644 PF14200 Ricin B 684 786Lectin 2 APG00537 Seq ID 27 PF03945 Endotoxin N 72 296 PF00555 EndotoxinM 301 507 PF03944 Endotoxin C 517 655 APG00537 Seq ID 28 Alternate startPF03945 Endotoxin N 69 293 modified PF00555 Endotoxin M 298 504 PF03944Endotoxin C 514 652 APG00537 Seq ID 29 Alternate start PF03945 EndotoxinN 69 293 modified and 3′ PF00555 Endotoxin M 298 504 Truncation PF03944Endotoxin C 514 651 APG00537 Seq ID 30 no PFAM Split-Cry C- domains termAPG00543 Seq ID 31 PF03945 Endotoxin N 76 304 PF03944 Endotoxin C 523682 APG00543 Seq ID 32 3′ Truncation PF03945 Endotoxin N 76 304 modifiedPF03944 Endotoxin C 523 681 APG00555 Seq ID 33 PF03945 Endotoxin N 80300 PF00555 Endotoxin M 305 515 PF03944 Endotoxin C 525 659 APG00555 SeqID 34 Alternate start PF03945 Endotoxin N 77 297 modified PF00555Endotoxin M 302 512. PF03944 Endotoxin C 522 656 APG00555 Seq ID 35Alternate start PF03945 Endotoxin N 77 297 modified and 3′ PF00555Endotoxin M 302 512 Truncation PF03944 Endotoxin C 522 655 APG00557 SeqID 36 PF05431 Toxin 10 210 404 APG00557 Seq ID 37 Alternate startPF05431 Toxin 10 201 395 modified APG00558 Seq ID 38 PF03945 Endotoxin N75 297 PF00555 Endotoxin M 302 521 PF03944 Endotoxin C 531 664 APG00558Seq ID 39 Alternate start PF03945 Endotoxin N 75 297 modified PF00555Endotoxin M 302 521 PF03944 Endotoxin C 531 664 APG00558 Seq ID 40Alternate start PF03945 Endotoxin N 75 297 modified and 3′ PF00555Endotoxin M 302 521 Truncation PF03944 Endotoxin C 531 663 APG00565 SeqID 41 PF03945 Endotoxin N 85 321 PF00555 Endotoxin M 326 532 PF03944Endotoxin C 542 691 APG00565 Seq ID 42 Alternate start PF03945 EndotoxinN 68 304 modified and 3′ PF00555 Endotoxin M 309 515 Truncation PF03944Endotoxin C 525 673 APG00565 Seq ID 43 Alternate start PF03945 EndotoxinN 68 304 modified PF00555 Endotoxin M 309 515 PF03944 Endotoxin C 525674 APG00566 Seq ID 44 PF03318 ETX MTX2 36 260 APG00566 Seq ID 45Alternate start PF03318 ETX MTX2 29 253 modified APG00572 Seq ID 46PF00388 PI-PLC-X 334 472 PF14200 Ricin B 760 866 Lectin 2 APG00587 SeqID 47 PF03945 Endotoxin N 111 312 PF14200 Ricin B 433 540 Lectin 2PF02839 CBM 5 12 557 593 APG00587 Seq ID 48 Signal peptide PF03945Endotoxin N 73 274 modified removed PF14200 Ricin B 395 502 Lectin 2PF02839 CBM 5 12 519 555 APG00939 Seq ID 49 PF12495 Vip3A N 16 188PF02018 CBM 4 9 546 670 PF02018 CBM 4 9 825 910 APG00939 Seq ID 50Alternate start PF12495 Vip3A N 14 186 modified PF02018 CBM 4 9 544 668PF02018 CBM 4 9 823 908 APG00606 Seq ID 51 PF03945 Endotoxin N 72 296PF00555 Endotoxin M 301 507 PF03944 Endotoxin C 517 649 APG00606 Seq ID52 Alternate start PF03945 Endotoxin N 69 293 modified and 3′ PF00555Endotoxin M 298 504 Truncation PF03944 Endotoxin C 514 645 APG00606 SeqID 53 Alternate start PF03945 Endotoxin N 69 293 modified PF00555Endotoxin M 298 504 PF03944 Endotoxin C 514 646 APG00607 Seq ID 54PF03945 Endotoxin N 37 232 APG00608 Seq ID 55 PF03945 Endotoxin N 76 341PF03944 Endotoxin C 572 727 APG00608 Seq ID 56 3′ Truncation PF03945Endotoxin N 76 341 modified PF03944 Endotoxin C 572 726 APG00609 Seq ID57 PF03318 ETX MTX2 66 327 APG00622 Seq ID 58 PF03945 Endotoxin N 69 161PF03945 Endotoxin N 188 335 PF03944 Endotoxin C 556 694 APG00622 Seq ID59 3′ Truncation PF03945 Endotoxin N 69 161 modified PF03945 Endotoxin N188 335 PF03944 Endotoxin C 556 693 APG00624 Seq ID 60 PF01338 Bac thurtoxin 65 297 PF14200 Ricin B 325 427 Lectin 2 PF14200 Ricin B 467 570Lectin 2 APG00624 Seq ID 61 Alternate start PF01338 Bac thur toxin 12244 modified PF14200 Ricin B 272 374 Lectin 2 PF14200 Ricin B 414 517Lectin 2 APG00637 Seq ID 62 PF03945 Endotoxin N 71 284 PF03945 EndotoxinN 290 360 PF00555 Endotoxin M 365 583 PF03944 Endotoxin C 593 725APG00637 Seq ID 63 Alternate start PF03945 Endotoxin N 66 280 modifiedand 3′ PF03945 Endotoxin N 284 355 Truncation PF00555 Endotoxin M 360578 PF03944 Endotoxin C 588 719 APG00637 Seq ID 64 Alternate startPF03945 Endotoxin N 66 279 modified PF03945 Endotoxin N 285 355 PF00555Endotoxin M 360 578 PF03944 Endotoxin C 588 720 APG00638 Seq ID 65PF00388 PI-PLC-X 78 204 PF14200 Ricin B 391 493 Lectin 2 APG00641 Seq ID66 PF03318 ETX MTX2 30 280 APG00641 Seq ID 67 Alternate start PF03318ETX MTX2 25 275 modified APG00643 Seq ID 68 PF03945 Endotoxin N 68 294PF00555 Endotoxin M 299 506 PF03944 Endotoxin C 516 647 APG00643 Seq ID69 3′ Truncation PF03945 Endotoxin N 68 294 modified PF00555 Endotoxin M299 506 PF03944 Endotoxin C 516 646 APG00644 Seq ID 70 PF03945 EndotoxinN 98 294 PF01473 CW binding 1 588 607 PF01473 CW binding 1 612 627PF01473 CW binding 1 655 670 PF01473 CW binding 1 816 831 APG00644 SeqID 71 Alternate start PF03945 Endotoxin N 93 289 modified PF01473 CWbinding 1 583 602 PF01473 CW binding 1 607 622 PF01473 CW binding 1 650665 PF01473 CW binding 1 811 826 APG00644 Seq ID 72 Signal peptidePF03945 Endotoxin N 55 251 modified removed PF01473 CW binding 1 545 564PF01473 CW binding 1 569 584 PF01473 CW binding 1 612 627 PF01473 CWbinding 1 773 788 APG00648 Seq ID 73 PF05431 Toxin 10 202 398 APG00648Seq ID 74 Signal peptide PF05431 Toxin 10 168 364 modified removedAPG00649 Seq ID 75 PF00652 Ricin B 40 170 Lectin PF05431 Toxin 10 181380 APG00649 Seq ID 76 Alternate start PF00652 Ricin B 38 168 modifiedLectin PF05431 Toxin 10 179 378 APG00651 Seq ID 77 PF03945 Endotoxin N65 315 PF00555 Endotoxin M 320 515 PF03944 Endotoxin C 533 673 APG00651Seq ID 78 3′ Truncation PF03945 Endotoxin N 65 315 modified PF00555Endotoxin M 320 515 PF03944 Endotoxin C 533 672 APG00657 Seq ID 79PF12495 Vip3A N 16 188 PF02018 CBM 4 9 814 914 APG00657 Seq ID 80Alternate start PF12495 Vip3A N 14 186 modified PF02018 CBM 4 9 812 912APG00659 Seq ID 81 no PFAM domains APG00659 Seq ID 82 Alternate start noPFAM modified domains APG00661 Seq ID 83 no PFAM domains APG00662 Seq ID84 PF03945 Endotoxin N 70 293 PF00555 Endotoxin M 298 504 PF03944Endotoxin C 515 651 APG00662 Seq ID 85 Alternate start PF03945 EndotoxinN 61 284 modified PF00555 Endotoxin M 289 495 PF03944 Endotoxin C 506642 APG00662 Seq ID 86 Alternate start PF03945 Endotoxin N 61 284modified and 3′ PF00555 Endotoxin M 289 495 Truncation PF03944 EndotoxinC 506 641 APG00663 Seq ID 87 PF03945 Endotoxin N 70 318 PF03944Endotoxin C 537 687 APG00663 Seq ID 88 3′ Truncation PF03945 Endotoxin N70 318 modified PF03944 Endotoxin C 537 686 APG00664 Seq ID 89 PF03945Endotoxin N 74 325 PF00555 Endotoxin M 332 531 PF03944 Endotoxin C 541687 APG00664 Seq ID 90 3′ Truncation PF03945 Endotoxin N 74 325 modifiedPF00555 Endotoxin M 332 531 PF03944 Endotoxin C 541 686 APG00672 Seq ID91 PF03945 Endotoxin N 98 335 PF03944 Endotoxin C 526 661 PF01473 CWbinding 1 685 702 PF01473 CW binding 1 714 731 PF01473 CW binding 1 743760 PF01473 CW binding 1 772 789 PF01473 CW binding 1 801 818 APG00672Seq ID 92 3′ Truncation PF03945 Endotoxin N 98 335 modified PF03944Endotoxin C 526 660 APG00673 Seq ID 93 PF03945 Endotoxin N 92 331PF00555 Endotoxin M 336 556 PF03944 Endotoxin C 567 719 APG00673 Seq ID94 Alternate start PF03945 Endotoxin N 62 302 modified and 3′ PF00555Endotoxin M 307 527 Truncation PF03944 Endotoxin C 538 689 APG00673 SeqID 95 Alternate start PF03945 Endotoxin N 63 302 modified PF00555Endotoxin M 307 527 PF03944 Endotoxin C 538 690 APG00674 Seq ID 96PF00652 Ricin B 8 143 Lectin PF05431 Toxin 10 152 349 APG00675 Seq ID 97PF12495 Vip3A N 10 187 APG00677 Seq ID 98 PF03945 Endotoxin N 66 299PF00555 Endotoxin M 304 505 PF03944 Endotoxin C 515 651 APG00679 Seq ID99 PF03945 Endotoxin N 35 253 PF00555 Endotoxin M 258 450 PF03944Endotoxin C 460 594 APG00679 Seq ID 100 3′ Truncation PF03945 EndotoxinN 35 253 modified PF00555 Endotoxin M 258 450 PF03944 Endotoxin C 460593 APG00687 Seq ID 101 PF03945 Endotoxin N 59 322 PF00555 Endotoxin M330 489 PF03944 Endotoxin C 530 667 APG00687 Seq ID 102 3′ TruncationPF03945 Endotoxin N 59 322 modified PF00555 Endotoxin M 330 490 PF03944Endotoxin C 530 666 APG00687 Seq ID 103 PF07029 CryBP1 1 119 CryBP1APG00688 Seq ID 104 PF03945 Endotoxin N 65 300 PF00555 Endotoxin M 305517 PF03944 Endotoxin C 527 665 APG00688 Seq ID 105 3′ TruncationPF03945 Endotoxin N 65 300 modified PF00555 Endotoxin M 305 517 PF03944Endotoxin C 527 664 APG00693 Seq ID 106 PF03318 ETX MTX2 91 318 APG00693Seq ID 107 Signal peptide PF03318 ETX MTX2 12 268 modified removedAPG00693 Seq ID 108 Alternate start PF03318 ETX MTX2 35 294 modifiedAPG00695 Seq ID 109 PF03945 Endotoxin N 61 295 PF00555 Endotoxin M 300494 PF03944 Endotoxin C 504 631 APG00695 Seq ID 110 Alternate startPF03945 Endotoxin N 58 292 modified and 3′ PF00555 Endotoxin M 297 491Truncation PF03944 Endotoxin C 501 627 APG00695 Seq ID 111 Alternatestart PF03945 Endotoxin N 58 292 modified PF00555 Endotoxin M 297 491PF03944 Endotoxin C 501 628 APG00695 Seq ID 112 no PFAM Split-Cry C-domains term APG00701 Seq ID 113 PF05431 Toxin 10 189 382 APG00701 SeqID 114 Signal peptide PF05431 Toxin 10 160 353 modified removed APG00702Seq ID 115 PF03945 Endotoxin N 109 309 PF14200 Ricin B 487 589 Lectin 2APG00702 Seq ID 116 Signal peptide PF03945 Endotoxin N 71 271 modifiedremoved PF14200 Ricin B 449 551 Lectin 2 APG00703 Seq ID 117 PF03945Endotoxin N 214 352 PF03944 Endotoxin C 573 723 APG00703 Seq ID 118Alternate start PF03945 Endotoxin N 208 346 modified PF03944 Endotoxin C567 717 APG00703 Seq ID 119 Alternate start PF03945 Endotoxin N 208 346modified and 3′ PF03944 Endotoxin C 567 716 Truncation APG00705 Seq ID120 PF03945 Endotoxin N 100 343 PF03944 Endotoxin C 535 672 APG00705 SeqID 121 Alternate start PF03945 Endotoxin N 94 337 modified PF03944Endotoxin C 529 666 APG00705 Seq ID 122 Signal peptide PF03945 EndotoxinN 65 308 modified removed and 3′ PF03944 Endotoxin C 500 636 TruncationAPG00705 Seq ID 123 Signal peptide PF03945 Endotoxin N 65 308 modifiedremoved PF03944 Endotoxin C 500 637 APG00705 Seq ID 124 Alternate startPF03945 Endotoxin N 94 337 modified and 3′ PF03944 Endotoxin C 529 665Truncation APG00705 Seq ID 125 3′ Truncation PF03945 Endotoxin N 100 343modified PF03944 Endotoxin C 535 671 APG00706 Seq ID 126 PF05431 Toxin10 251 446 APG00707 Seq ID 127 PF03318 ETX MTX2 20 277 APG00707 Seq ID128 Signal peptide PF03318 ETX MTX2 9 259 modified removed APG00710 SeqID 129 PF03945 Endotoxin N 86 336 PF00555 Endotoxin M 343 533 PF03944Endotoxin C 543 679 APG00710 Seq ID 130 Alternate start PF03945Endotoxin N 63 313 modified PF00555 Endotoxin M 320 510 PF03944Endotoxin C 520 656 APG00710 Seq ID 131 Alternate start PF03945Endotoxin N 63 313 modified and 3′ PF00555 Endotoxin M 320 510Truncation PF03944 Endotoxin C 520 655 APG00718 Seq ID 132 PF03318 ETXMTX2 34 292 APG00721 Seq ID 133 PF03945 Endotoxin N 37 270 PF03945Endotoxin N 299 346 PF00555 Endotoxin M 351 462 PF03944 Endotoxin C 592729 APG00721 Seq ID 134 3′ Truncation PF03945 Endotoxin N 37 270modified PF03945 Endotoxin N 299 346 PF00555 Endotoxin M 351 463 PF03944Endotoxin C 592 728 APG00721 Seq ID 135 no PFAM Split-Cry C- domainsterm APG00722 Seq ID 136 PF14200 Ricin B 45 146 Lectin 2 PF05431 Toxin10 152 348 APG00724 Seq ID 137 PF05431 Toxin 10 209 402 APG00724 Seq ID138 Signal peptide PF05431 Toxin 10 182 375 modified removed APG00726Seq ID 139 PF03945 Endotoxin N 86 339 PF00555 Endotoxin M 350 559PF03944 Endotoxin C 569 711 APG00726 Seq ID 140 Alternate start PF03945Endotoxin N 72 325 modified PF00555 Endotoxin M 336 545 PF03944Endotoxin C 555 697 APG00726 Seq ID 141 Alternate start PF03945Endotoxin N 72 325 modified and 3′ PF00555 Endotoxin M 336 545Truncation PF03944 Endotoxin C 555 696 APG00729 Seq ID 142 PF05431 Toxin10 184 385 APG00729 Seq ID 143 Signal peptide PF05431 Toxin 10 157 358modified removed APG00735 Seq ID 144 PF14200 Ricin B 69 172 Lectin 2PF05431 Toxin 10 178 375 APG00735 Seq ID 145 Alternate start PF14200Ricin B 43 146 modified Lectin 2 PF05431 Toxin 10 152 349 APG00781 SeqID 146 PF03945 Endotoxin N 59 294 PF00555 Endotoxin M 305 509 PF03944Endotoxin C 519 656 PF00652 Ricin B 672 801 Lectin APG00781 Seq ID 1473′ Truncation PF03945 Endotoxin N 59 294 modified PF00555 Endotoxin M305 509 PF03944 Endotoxin C 519 652 APG00784 Seq ID 148 PF03945Endotoxin N 67 316 PF03944 Endotoxin C 531 673 APG00784 Seq ID 149 3′Truncation PF03945 Endotoxin N 67 316 modified PF03944 Endotoxin C 531672 APG00785 Seq ID 150 PF03945 Endotoxin N 72 302 PF00555 Endotoxin M314 501 PF03944 Endotoxin C 511 670 APG00785 Seq ID 151 Alternate startPF03945 Endotoxin N 69 299 modified PF00555 Endotoxin M 311 498 PF03944Endotoxin C 508 667 APG00785 Seq ID 152 Alternate start PF03945Endotoxin N 69 299 modified and 3′ PF00555 Endotoxin M 311 498Truncation PF03944 Endotoxin C 508 666 APG00786 Seq ID 153 PF03945Endotoxin N 67 288 PF00555 Endotoxin M 293 505 PF03944 Endotoxin C 516653 APG00786 Seq ID 154 Alternate start PF03945 Endotoxin N 61 282modified and 3′ PF00555 Endotoxin M 287 499 Truncation PF03944 EndotoxinC 510 646 APG00786 Seq ID 155 Alternate start PF03945 Endotoxin N 61 282modified PF00555 Endotoxin M 287 499 PF03944 Endotoxin C 510 647APG00787 Seq ID 156 PF03945 Endotoxin N 110 311 PF01473 CW binding 1 534550 APG00787 Seq ID 157 Signal peptide PF03945 Endotoxin N 72 274modified removed PF01473 CW binding 1 496 512 APG00799 Seq ID 158PF03945 Endotoxin N 63 260 PF03944 Endotoxin C 463 630 APG00799 Seq ID159 3′ Truncation PF03945 Endotoxin N 63 260 modified PF03944 EndotoxinC 463 629 APG00801 Seq ID 160 PF03945 Endotoxin N 67 316 PF03944Endotoxin C 527 666 APG00801 Seq ID 161 3′ Truncation PF03945 EndotoxinN 67 316 modified PF03944 Endotoxin C 527 665 APG00802 Seq ID 162PF03945 Endotoxin N 65 285 PF00555 Endotoxin M 295 498 PF03944 EndotoxinC 508 653 APG00802 Seq ID 163 3′ Truncation PF03945 Endotoxin N 65 285modified PF00555 Endotoxin M 295 498 PF03944 Endotoxin C 508 652APG00805 Seq ID 164 PF03945 Endotoxin N 75 296 PF00555 Endotoxin M 301500 PF03944 Endotoxin C 511 643 APG00805 Seq ID 165 Alternate startPF03945 Endotoxin N 72 293 modified and 3′ PF00555 Endotoxin M 298 497Truncation PF03944 Endotoxin C 508 639 APG00805 Seq ID 166 Alternatestart PF03945 Endotoxin N 72 293 modified PF00555 Endotoxin M 298 497PF03944 Endotoxin C 508 640 APG00806 Seq ID 167 PF05431 Toxin 10 203 396APG00806 Seq ID 168 Signal peptide PF00652 Ricin B 5 100 modifiedremoved Lectin PF05431 Toxin 10 174 367 APG00807 Seq ID 169 PF03318 ETXMTX2 32 278 APG00807 Seq ID 170 Alternate start PF03318 ETX MTX2 30 276modified APG00810 Seq ID 171 PF03945 Endotoxin N 62 286 PF00555Endotoxin M 291 489 PF03944 Endotoxin C 499 635 APG00810 Seq ID 172Alternate start PF03945 Endotoxin N 59 283 modified and 3′ PF00555Endotoxin M 288 486 Truncation PF03944 Endotoxin C 496 631 APG00810 SeqID 173 Alternate start PF03945 Endotoxin N 59 283 modified PF00555Endotoxin M 288 486 PF03944 Endotoxin C 496 632 APG00864 Seq ID 174PF03945 Endotoxin N 74 327 PF00555 Endotoxin M 332 526 PF03944 EndotoxinC 543 683 APG00912 Seq ID 175 PF03945 Endotoxin N 70 308 PF00555Endotoxin M 313 530 PF03944 Endotoxin C 540 678 APG00912 Seq ID 176Alternate start PF03945 Endotoxin N 64 302 modified PF00555 Endotoxin M307 524 PF03944 Endotoxin C 534 672 APG00912 Seq ID 177 Alternate startPF03945 Endotoxin N 64 302 modified and 3′ PF00555 Endotoxin M 307 524Truncation PF03944 Endotoxin C 534 671 APG00912 Seq ID 178 PF07029CryBP1 37 195 CryBP1 APG00960 Seq ID 179 PF03945 Endotoxin N 51 277PF00555 Endotoxin M 282 491 PF03944 Endotoxin C 501 644 APG00960 Seq ID180 Alternate start PF03945 Endotoxin N 36 262 modified and 3′ PF00555Endotoxin M 267 476 Truncation PF03944 Endotoxin C 486 628 APG00960 SeqID 181 Alternate start PF03945 Endotoxin N 36 262 modified PF00555Endotoxin M 267 476 PF03944 Endotoxin C 486 629 APG00972 Seq ID 182PF03945 Endotoxin N 91 326 PF00555 Endotoxin M 332 540 PF03944 EndotoxinC 550 704 APG00972 Seq ID 183 Alternate start PF03945 Endotoxin N 68 303modified and 3′ PF00555 Endotoxin M 309 517 Truncation PF03944 EndotoxinC 527 680 APG00972 Seq ID 184 Alternate start PF03945 Endotoxin N 68 303modified PF00555 Endotoxin M 309 517 PF03944 Endotoxin C 527 681APG00980 Seq ID 185 PF03318 ETX MTX2 94 315 APG00980 Seq ID 186 Signalpeptide PF03318 ETX MTX2 44 265 modified removed APG00980 Seq ID 187Alternate start PF03318 ETX MTX2 77 298 modified APG00981 Seq ID 188PF03945 Endotoxin N 71 328 PF00555 Endotoxin M 333 525 PF03944 EndotoxinC 535 675 APG00981 Seq ID 189 3′ Truncation PF03945 Endotoxin N 71 328modified PF00555 Endotoxin M 333 525 PF03944 Endotoxin C 535 674APG00986 Seq ID 190 PF03945 Endotoxin N 76 305 PF00555 Endotoxin M 313488 PF03944 Endotoxin C 499 635 APG00986 Seq ID 191 Alternate startPF03945 Endotoxin N 73 302 modified PF00555 Endotoxin M 310 485 PF03944Endotoxin C 496 632 APG00986 Seq ID 192 Alternate start PF03945Endotoxin N 73 302 modified and 3′ PF00555 Endotoxin M 310 485Truncation PF03944 Endotoxin C 496 631 APG00986 Seq ID 193 no PFAMSplit-Cry C- domains term APG00988 Seq ID 194 PF05431 Toxin 10 190 386APG00988 Seq ID 195 Signal peptide PF05431 Toxin 10 163 359 modifiedremoved APG01000 Seq ID 196 PF05431 Toxin 10 57 251 APG01000 Seq ID 197Alternate start PF05431 Toxin 10 39 233 modified APG01003 Seq ID 198PF12495 Vip3A N 16 188 PF02018 CBM 4 9 545 666 APG01003 Seq ID 199Alternate start PF12495 Vip3A N 14 186 modified PF02018 CBM 4 9 543 664APG01028 Seq ID 200 PF03945 Endotoxin N 80 310 PF00555 Endotoxin M 315529 PF03944 Endotoxin C 539 671 APG01028 Seq ID 201 Alternate startPF03945 Endotoxin N 77 307 modified PF00555 Endotoxin M 312 526 PF03944Endotoxin C 536 668 APG01028 Seq ID 202 Alternate start PF03945Endotoxin N 77 307 modified and 3′ PF00555 Endotoxin M 312 526Truncation PF03944 Endotoxin C 536 667 APG01028 Seq ID 203 no PFAMSplit-Cry C- domains term APG01112 Seq ID 204 PF03945 Endotoxin N 2 200APG00556 Seq ID 205 no PFAM domains APG00556 Seq ID 206 Alternate startno PFAM modified domains APG00623 Seq ID 207 no PFAM domains APG01037Seq ID 208 no PFAM domains APG01037.1 Seq ID 209 Alternate start no PFAM(APG01037 domains modified) APG01037.4 Seq ID 210 Alternate start noPFAM (APG01037 and point domains modified) mutation (S to T) APG01037.5Seq ID 211 Alternate start no PFAM (APG01037 and point domains modified)mutation (S to T) APG01037.6 Seq ID 212 Alternate start no PFAM(APG01037 and point domains modified) mutation (S to K) APG01037.7 SeqID 213 Alternate start no PFAM (APG01037 and point domains modified)mutation (T to E) APG01037.8 Seq ID 214 Alternate start no PFAM(APG01037 and point domains modified) mutation (I to E) APG01086 Seq ID215 PF03318 ETX MTX2 34 260 APG01086 Seq ID 216 Alternate start PF03318ETX MTX2 28 253 modified APG06508 Seq ID 217 no PFAM domains APG09801Seq ID 218 no PFAM domains

ii. Variants and Fragments of Pesticidal Proteins and PolynucleotidesEncoding the Same

Pesticidal proteins or polypeptides of the invention include those setforth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, and/or 218 and fragments and variants thereof. By “pesticidaltoxin” or “pesticidal protein” or “pesticidal polypeptide” is intended atoxin or protein or polypeptide that has activity against one or morepests, including, insects, fungi, nematodes, and the like such that thepest is killed or controlled.

An “isolated” or “purified” polypeptide or protein, or biologicallyactive portion thereof, is substantially or essentially free fromcomponents that normally accompany or interact with the polypeptide orprotein as found in its naturally occurring environment. Thus, anisolated or purified polypeptide or protein is substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. A protein that is substantiallyfree of cellular material includes preparations of protein having lessthan about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminatingprotein. When the protein of the invention or biologically activeportion thereof is recombinantly produced, optimally culture mediumrepresents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) ofchemical precursors or non-protein-of-interest chemicals.

The term “fragment” refers to a portion of a polypeptide sequence of theinvention. “Fragments” or “biologically active portions” includepolypeptides comprising a sufficient number of contiguous amino acidresidues to retain the biological activity, i.e., have pesticidalactivity. Fragments of the pesticidal proteins include those that areshorter than the full-length sequences, either due to the use of analternate downstream start site, or due to processing that produces ashorter protein having pesticidal activity. Processing may occur in theorganism the protein is expressed in, or in the pest after ingestion ofthe protein. Examples of fragments of the proteins can be found inTable 1. A biologically active portion of a pesticidal protein can be apolypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250 or moreamino acids in length of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, and/or 218. Such biologically activeportions can be prepared by recombinant techniques and evaluated forpesticidal activity. As used here, a fragment comprises at least 8contiguous amino acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, and/or 218.

Bacterial genes, including those encoding the pesticidal proteinsdisclosed herein, quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. These start codons can include ATGcodons. However, bacteria such as Bacillus sp. also recognize the codonGTG as a start codon, and proteins that initiate translation at GTGcodons contain a methionine at the first amino acid. On rare occasions,translation in bacterial systems can initiate at a TTG codon, though inthis event the TTG encodes a methionine. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may also lead to generation of pesticidal proteins.These pesticidal proteins are encompassed in the present invention andmay be used in the methods disclosed herein. It will be understood that,when expressed in plants, it will be necessary to alter the alternatestart codon to ATG for proper translation.

In various embodiments the pesticidal proteins provided herein includeamino acid sequences deduced from the full-length nucleotide sequencesand amino acid sequences that are shorter than the full-length sequencesdue to the use of an alternate downstream start site. Thus, thenucleotide sequence of the invention and/or vectors, host cells, andplants comprising the nucleotide sequence of the invention (and methodsof making and using the nucleotide sequence of the invention) maycomprise a nucleotide sequence encoding an alternate start site.

It is recognized that modifications may be made to the pesticidalpolypeptides provided herein creating variant proteins. Changes designedby man may be introduced through the application of site-directedmutagenesis techniques. Alternatively, native, as yet-unknown or as yetunidentified polynucleotides and/or polypeptides structurally and/orfunctionally-related to the sequences disclosed herein may also beidentified that fall within the scope of the present invention.Conservative amino acid substitutions may be made in nonconservedregions that do not alter the function of the pesticidal proteins.Alternatively, modifications may be made that improve the activity ofthe toxin. Modification of Cry toxins by domain III swapping hasresulted in some cases in hybrid toxins with improved toxicities againstcertain insect species. Thus, domain III swapping could be an effectivestrategy to improve toxicity of Cry toxins or to create novel hybridtoxins with toxicity against pests that show no susceptibility to theparental Cry toxins. Site-directed mutagenesis of domain II loopsequences may result in new toxins with increased insecticidal activity.Domain II loop regions are key binding regions of initial Cry toxinsthat are suitable targets for the mutagenesis and selection of Crytoxins with improved insecticidal properties. Domain I of the Cry toxinmay be modified to introduce protease cleavage sites to improve activityagainst certain pests. Strategies for shuffling the three differentdomains among large numbers of cry genes and high through outputbioassay screening methods may provide novel Cry toxins with improved ornovel toxicities.

As indicated, fragments and variants of the polypeptides disclosedherein will retain pesticidal activity. Pesticidal activity comprisesthe ability of the composition to achieve an observable effectdiminishing the occurrence or an activity of the target pest, includingfor example, bringing about death of at least one pest, or a noticeablereduction in pest growth, feeding, or normal physiological development.Such decreases in numbers, pest growth, feeding or normal developmentcan comprise any statistically significant decrease, including, forexample a decrease of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95% or greater. The pesticidalactivity against one or more of the various pests provided herein,including, for example, pesticidal activity against Coleoptera, Diptera,Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera,Nematodes, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., or any other pest described herein. It is recognizedthat the pesticidal activity may be different or improved relative tothe activity of the native protein, or it may be unchanged, so long aspesticidal activity is retained. Methods for measuring pesticidalactivity are provide elsewhere herein. See also, Czapla and Lang (1990)J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J.252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293;and U.S. Pat. No. 5,743,477, all of which are herein incorporated byreference in their entirety.

By “variants” is intended polypeptides having an amino acid sequencethat is at least about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to the amino acid sequence of any ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,and/or 218 and retain pesticidal activity. Note, Table 1 providesnon-limiting examples of variant polypeptides (and polynucleotideencoding the same) for each of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, and/or 218. A biologically active variantof a pesticidal polypeptide of the invention may differ by as few asabout 1-15 amino acid residues, as few as about 1-10, such as about6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1amino acid residue. In specific embodiments, the polypeptides cancomprise an N-terminal or a C-terminal truncation, which can comprise atleast a deletion of 10, 15, 20, 25, 30, 35, 40, 45, 50 amino acids ormore from either the N or C terminal of the polypeptide.

Recombinant or synthetic nucleic acids encoding the pesticidalpolypeptides disclosed herein are also provided. Of particular interestare nucleic acid sequences that have been designed for expression in aplant of interest. That is, the nucleic acid sequence can be optimizedfor increased expression in a host plant. A pesticidal protein of theinvention can be back-translated to produce a nucleic acid comprisingcodons optimized for expression in a particular host, for example, acrop plant. In another embodiment, the polynucleotides encoding thepolypeptides provided herein may be optimized for increased expressionin the transformed plant. That is, the polynucleotides can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference. Expression of such acoding sequence by the transformed plant (e.g., dicot or monocot) willresult in the production of a pesticidal polypeptide and conferincreased resistance in the plant to a pest. Recombinant and syntheticnucleic acid molecules encoding the pesticidal proteins of the inventiondo not include the naturally occurring bacterial sequence encoding theprotein.

A “recombinant polynucleotide” or “recombinant nucleic acid” comprises acombination of two or more chemically linked nucleic acid segments whichare not found directly joined in nature. By “directly joined” isintended the two nucleic acid segments are immediately adjacent andjoined to one another by a chemical linkage. In specific embodiments,the recombinant polynucleotide comprises a polynucleotide of interest ora variant or fragment thereof such that an additional chemically linkednucleic acid segment is located either 5′, 3′ or internal to thepolynucleotide of interest. Alternatively, the chemically-linked nucleicacid segment of the recombinant polynucleotide can be formed by deletionof a sequence. The additional chemically linked nucleic acid segment orthe sequence deleted to join the linked nucleic acid segments can be ofany length, including for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20or greater nucleotides. Various methods for making such recombinantpolynucleotides include chemical synthesis or by the manipulation ofisolated segments of polynucleotides by genetic engineering techniques.In specific embodiments, the recombinant polynucleotide can comprise arecombinant DNA sequence or a recombinant RNA sequence. A “fragment of arecombinant polynucleotide or nucleic acid” comprises at least one of acombination of two or more chemically linked amino acid segments whichare not found directly joined in nature.

Fragments of a polynucleotide (RNA or DNA) may encode protein fragmentsthat retain activity. In specific embodiments, a fragment of arecombinant polynucleotide or a recombinant polynucleotide constructcomprises at least one junction of the two or more chemically linked oroperably linked nucleic acid segments which are not found directlyjoined in nature. A fragment of a polynucleotide that encodes abiologically active portion of a polypeptide that retains pesticidalactivity will encode at least 25, 30, 40, 50, 60, 70, 75, 80, 90, 100,110, 120, 125, 130, 140, 150, 160, 170, 175, 180, contiguous aminoacids, or up to the total number of amino acids present in a full-lengthpolypeptide as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, and/or 218. In specific embodiments, suchpolypeptide fragments are active fragment, and in still otherembodiments, the polypeptide fragment comprises a recombinantpolypeptide fragment. As used herein, a fragment of a recombinantpolypeptide comprises at least one of a combination of two or morechemically linked amino acid segments which are not found directlyjoined in nature.

By “Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, an isolated polynucleotide thatencodes a polypeptide with a given percent sequence identity to thepolypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216, 217, and/or 218 are disclosed. Percent sequence identity betweenany two polypeptides can be calculated using sequence alignment programsand parameters described elsewhere herein. Where any given pair ofpolynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, and/or 218.

Variant polynucleotide and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different pesticidalprotein disclosed herein (SEQ ID NO: 1-218) is manipulated to create anew pesticidal protein possessing the desired properties. In thismanner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequenceregions that have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. For example, using this approach,sequence motifs encoding a domain of interest may be shuffled betweenthe pesticidial sequences provided herein and other known pesticidialgenes to obtain a new gene coding for a protein with an improvedproperty of interest, such as an increased K_(m) in the case of anenzyme. Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458. A“shuffled” nucleic acid is a nucleic acid produced by a shufflingprocedure such as any shuffling procedure set forth herein. Shufflednucleic acids are produced by recombining (physically or virtually) twoor more nucleic acids (or character strings), for example in anartificial, and optionally recursive, fashion. Generally, one or morescreening steps are used in shuffling processes to identify nucleicacids of interest; this screening step can be performed before or afterany recombination step. In some (but not all) shuffling embodiments, itis desirable to perform multiple rounds of recombination prior toselection to increase the diversity of the pool to be screened. Theoverall process of recombination and selection are optionally repeatedrecursively. Depending on context, shuffling can refer to an overallprocess of recombination and selection, or, alternately, can simplyrefer to the recombinational portions of the overall process.

In one embodiment, a method of obtaining a polynucleotide that encodesan improved polypeptide comprising pesticidal activity is provided,wherein the improved polypeptide has at least one improved property overany one of SEQ ID NOS: 1-218. Such methods can comprises (a) recombininga plurality of parental polynucleotides to produce a library ofrecombinant polynucleotides encoding recombinant pesticidalpolypeptides; (b) screening the library to identify a recombinantpolynucleotide that encodes an improved recombinant pesticidalpolypeptide that has an enhanced property improved over the parentalpolynucleotide; (c) recovering the recombinant polynucleotide thatencodes the improved recombinant pesticidal polypeptide identified in(b); and, (d) repeating steps (a), (b) and (c) using the recombinantpolynucleotide recovered in step (c) as one of the plurality of parentalpolynucleotides in repeated step (a).

Provided herein are active variants of the polypeptides set forth in SEQID NOS: 205, 206, 207, 208, 209, 210, 211, 212, 213, and/or 214. Suchpolypeptides comprise a sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to any one of SEQ ID NOS: 205, 206, 207, 208, 209, 210, 211,212, 213, and/or 214 and further comprises one or more of themodifications set forth in Table 3. Any given variant of SEQ ID NO: 205,206, 207, 208, 209, 210, 211, 212, 213, and/or 214 can have one or moreof any combination of amino acid alterations in the corresponding aminoacid position(s) as set forth in Table 3, or fragments thereof. Such,variants will retain pesticidal activity. In specific embodiments, suchvariants will have improved pesticidal activity against an insect ofinterest.

Further provided are polypeptides comprising a sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to any one of SEQ ID NOS: 205, 206, 207,208, 209, 210, 211, 212, 213, and/or 214 and further comprise at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the modifications set forth inTable 3, wherein the amino acid positions 204-210 corresponding to thepositions of SEQ ID NO:208 (DHYFWFL) are not altered.

Table 3 provides protein variants of any one of SEQ ID NO: 205-214 asindicated by amino acid position and change. The amino acid positiondenoted in Table 3 reflects the amino acid position of SEQ ID NO:208(APG01037.0). Corresponding amino acid positions in SEQ ID NO: 205-207and 209-214 can be determined using methods discussed elsewhere herein.

TABLE 3 Gene name AA position and change APG01037.1 K6L, V APG01037.1F7T APG01037.1 S10T APG01037.1 E11D APG01037.1 V15L APG01037.1 G16DAPG01037.1 N18T APG01037.1 P19T APG01037.1 N20D APG01037.1 F26DAPG01037.1. E28D APG01037.1 R29L APG01037.1 F30Y APG01037.1 Y44FAPG01037.1 Y45F APG01037.1 N46D APG01037.1 Q55R APG01037.1 T63MAPG01037.1 E66P APG01037.1 T68I APG01037.1 Y69F APG01037.1 Q70RAPG01037.1 Q75N APG01037.1 P77G APG01037.1 S78N APG01037.1 I81FAPG01037.1 N89Q APG01037.1 H90P APG01037.1 S96E APG01037.1 G103EAPG01037.1 N104Q APG01037.1 Q114K, Y, F, E APG01037.1 K124D APG01037.1T126I APG01037.1 L127E APG01037.1 V130F APG01037.1 F137Y APG01037.1S138N APG01037.1 V139F APG01037.1 S147N APG01037.1 T149E APG01037.1S157P APG01037.1 V159Q APG01037.1 T160S APG01037.1 N162K APG01037.1K168Q APG01037.1 K169R, M APG01037.1 K170E, Y, T APG01037.1 M177K, RAPG01037.1 Q179K APG01037.1 M182G, L, V, I APG01037.1 N183Q APG01037.1Q185R, K APG01037.1 Q191N APG01037.1 S193M APG01037.1 F194Y APG01037.1R200K APG01037.1 K201R APG01037.1 V202A, I APG01037.1 E203Q, DAPG01037.1 D213E APG01037.1 N214F APG01037.1 S218K APG01037.1 T220DAPG01037.1 T222P APG01037.1 K228N APG01037.1 F233Y

The amino acid position denoted in Table 3 reflects the amino acidposition of SEQ ID NO:208 (APG01037.0). The amino acid alterations setforth in Table 3 can be made in the corresponding amino acid positionsof any one of SEQ ID NOs: 205, 206, 207, 208, 209, 210, 211, 212, 213,and/or 214. With respect to an amino acid sequence that is optimallyaligned with a reference sequence, an amino acid residue “correspondsto” the position in the reference sequence with which the residue ispaired in the alignment. The “position” is denoted by a number thatsequentially identifies each amino acid in the reference sequence basedon its position relative to the N-terminus. For example, in SEQ ID NO:208 position 1 is L, position 2 is M, position 3 is P, etc. When a testsequence is optimally aligned with SEQ ID NO: 208, a residue in the testsequence that aligns with the P at position 3 is said to “correspond toposition 3” of SEQ ID NO: 208. Owing to deletions, insertion,truncations, fusions, etc., that must be taken into account whendetermining an optimal alignment, in general the amino acid residuenumber in a test sequence as determined by simply counting from theN-terminal will not necessarily be the same as the number of itscorresponding position in the reference sequence. For example, in a casewhere there is a deletion in an aligned test sequence, there will be noamino acid that corresponds to a position in the reference sequence atthe site of deletion. Where there is an insertion in an alignedreference sequence, that insertion will not correspond to any amino acidposition in the reference sequence. In the case of truncations orfusions there can be stretches of amino acids in either the reference oraligned sequence that do not correspond to any amino acid in thecorresponding sequence.

iii. Sequence Comparisons

As used herein, the term “identity” or “percent identity” when used withrespect to a particular pair of aligned amino acid sequences, refers tothe percent amino acid sequence identity that is obtained by countingthe number of identical matches in the alignment and dividing suchnumber of identical matches by the length of the aligned sequences. Asused herein, the term “similarity” or “percent similarity” when usedwith respect to a particular pair of aligned amino acid sequences,refers to the sum of the scores that are obtained from a scoring matrixfor each amino acid pair in the alignment divided by the length of thealigned sequences.

Unless otherwise stated, identity and similarity will be calculated bythe Needleman-Wunsch global alignment and scoring algorithms (Needlemanand Wunsch (1970) J. Mol. Biol. 48(3):443-453) as implemented by the“needle” program, distributed as part of the EMBOSS software package(Rice, P. Longden, I. and Bleasby, A., EMBOSS: The European MolecularBiology Open Software Suite, 2000, Trends in Genetics 16, (6) pp276-2′77, versions 6.3.1 available from EMBnet atembnet.org/resource/emboss and emboss.sourceforge.net, among othersources) using default gap penalties and scoring matrices (EBLOSUM62 forprotein and EDNAFULL for DNA). Equivalent programs may also be used. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide residue matches and an identical percent sequenceidentity when compared to the corresponding alignment generated byneedle from EMBOSS version 6.3.1.

Additional mathematical algorithms are known in the art and can beutilized for the comparison of two sequences. See, for example, thealgorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTprograms of Altschul et al. (1990) J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the BLASTN program (nucleotidequery searched against nucleotide sequences) to obtain nucleotidesequences homologous to pesticidal-like nucleic acid molecules of theinvention, or with the BLASTX program (translated nucleotide querysearched against protein sequences) to obtain protein sequenceshomologous to pesticidal nucleic acid molecules of the invention. BLASTprotein searches can be performed with the BLASTP program (protein querysearched against protein sequences) to obtain amino acid sequenceshomologous to pesticidal protein molecules of the invention, or with theTBLASTN program (protein query searched against translated nucleotidesequences) to obtain nucleotide sequences homologous to pesticidalprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. Alignment may also be performed manuallyby inspection.

Two sequences are “optimally aligned” when they are aligned forsimilarity scoring using a defined amino acid substitution matrix (e.g.,BLOSUM62), gap existence penalty and gap extension penalty so as toarrive at the highest score possible for that pair of sequences. Aminoacid substitution matrices and their use in quantifying the similaritybetween two sequences are well-known in the art and described, e.g., inDayhoff et al. (1978) “A model of evolutionary change in proteins.” In“Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3 (ed. M. O.Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. andHenikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919. TheBLOSUM62 matrix is often used as a default scoring substitution matrixin sequence alignment protocols. The gap existence penalty is imposedfor the introduction of a single amino acid gap in one of the alignedsequences, and the gap extension penalty is imposed for each additionalempty amino acid position inserted into an already opened gap. Thealignment is defined by the amino acids positions of each sequence atwhich the alignment begins and ends, and optionally by the insertion ofa gap or multiple gaps in one or both sequences, so as to arrive at thehighest possible score. While optimal alignment and scoring can beaccomplished manually, the process is facilitated by the use of acomputer-implemented alignment algorithm, e.g., gapped BLAST 2.0,described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, andmade available to the public at the National Center for BiotechnologyInformation Website (www.ncbi.nlm.nih.gov). Optimal alignments,including multiple alignments, can be prepared using, e.g., PSI-BLAST,available through www.ncbi.nlm.nih.gov and described by Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402.

iv. Antibodies

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; and U.S. Pat. No. 4,196,265). These antibodiescan be used in kits for the detection and isolation of toxinpolypeptides. Thus, this disclosure provides kits comprising antibodiesthat specifically bind to the polypeptides described herein, including,for example, polypeptides having the sequence of SEQ ID NOs: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, and/or 218.

II. Pests

The compositions and methods provided herein are useful against avariety of pests. “Pests” includes but is not limited to, insects,fungi, bacteria, nematodes, acarids, protozoan pathogens,animal-parasitic liver flukes, and the like. Pests of particularinterest are insect pests, particularly insect pests that causesignificant damage to agricultural plants. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, or nematodes. Innon-limiting embodiments, the insect pest comprises Western cornrootworm, Diabrotica virgifera virgifera; Fall armyworm, Spodopterafrugiperda; Colorado potato beetle, Leptinotarsa decemlineata; Cornearworm, Helicoverpa zea (in North America same species attacks cottonand called cotton bollworm); European corn borer, Ostrinia nubilalis;Black cutworm, Agrotis ipsilon; Diamondback moth, Plutella xylostella;Velvetbean caterpillar, Anticarsia gemmatalis; Southwestern corn borer,Diatraea grandiosella; Cotton bollworm, Helicoverpa armigera (foundother than USA in rest of the world); Southern green stinkbug, Nezaraviridula; Green stinkbug, Chinavia halaris; Brown marmorated stinkbug,Halyomorpha halys; and Brown stinbug, Euschistus servus Euschistus heros(Neotropical brown stink bug OR soy stink bug); Piezodorus guildinii(red-banded stink bug); Dichelops melacanthus (no common name) and/orDichelops furcatus (no common name); an aphid, such as a soybean aphid.In other embodiments, the pest comprises a nematode including, but notlimited to, Meloidogyne hapla (Northern root-knot nematode); Meloidogyneenterolobii, Meloidogyne arenaria (peanut root-knot nematode); andMeloidogyne javanica.

The term “insect pests” as used herein refers to insects and othersimilar pests such as, for example, those of the order Acari including,but not limited to, mites and ticks. Insect pests of the presentinvention include, but are not limited to, insects of the orderLepidoptera, e.g. Achoroia grisella, Acleris gloverana, Acleris variana,Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophilapometaria, Amyelois transitella, Anagasta kuehniella, Anarsialineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis,Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrixthurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Coliaseurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella,Datana integerrima, Dendrolimus sibericus, Desmiafeneralis, Diaphaniahyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraeasaccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia elutella,Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoelliaambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoamessoria, Galleria mellonella, Grapholita molesta, Harrisina americana,Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileucaoliviae, Homoeosoma electellum, Hyphantia cunea, Keiferialycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellarialugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis,Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestrabrassicae, Mamestra configurata, Manduca quinquemaculata, Manduca sexta,Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp.,Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes,Pectinophora gossypiella, Phryganidia californica, Phyllonorycterblancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynotaflouendanci, Platynota stultana, Platyptilia carduidactyla, Plodiainterpunctella, Plutella xylostella, Pontia protodice, Pseudaletiaunipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizuraconcinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp.,Thaurnstopoea pityocampa, Tinsola bisselliella, Trichoplusia hi, Udearubigalis, Xylomyges curiails, and Yponomeuta padella.

Insect pests also include insects selected from the orders Diptera,Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera,Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera,Coleoptera. Insect pests of the invention for the major crops include,but are not limited to: Maize: Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Helicoverpa zeae, corn earworm;Spodoptera frugiperda, fall armyworm; Diatraea grandiosella,southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalkborer; Diatraea saccharalis, surgarcane borer; western corn rootworm,e.g., Diabrotica virgifera virgifera; northern corn rootworm, e.g.,Diabrotica longicornis barberi; southern corn rootworm, e.g., Diabroticaundecimpunctata howardi; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blotch leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, two spotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, lesercornstalk borer; Feltia subterranea, granulate cutworm; Phyllophagacrinita, white grub; Eleodes, Conoderus, and Aeolus spp., wireworms;Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn fleabeetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; cornleaf aphid; Sipha flava, yellow sugarcane aphid; chinch bug, e.g.,Blissus leucopterus leucopterus; Contarinia sorghicola, sorghum midge;Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae,two-spotted spider mite; Wheat: Pseudaletia unipunctata, army worm;Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lessercornstalk borer; Agrotis orthogonia, pale western cutworm; Elasmopalpuslignosellus, lesser cornstalk borer; Oulema melanopus, cereal leafbeetle; Hypera punctata, clover leaf weevil; southern corn rootworm,e.g., Diabrotica undecimpunctata howardi; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Cylindrocupturus adspersus, sunflower stem weevil; Smicronyx fulus, redsunflower seed weevil; Smicronyx sordidus, gray sunflower seed weevil;Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; Zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, tobacco budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; boll weevil, e.g., Anthonomus grandis; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, two-spottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhoper;chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternum hilare,green stink bug; Soybean: Pseudoplusia includens, soybean looper;Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, greencloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon,black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens,tobacco budworm; Helicoverpa zea, cotton bollworm; Epilachna varivestis,Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae,potato leafhopper; Acrosternum hilare, green stink bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus differentialis,differential grasshopper; Hylemya platura, seedcorn maggot; Sericothripsvariabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychusturkestani, strawberry spider mite; Tetranychus urticae, two-spottedspider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotisipsilon, black cutworm; Schizaphis graminum, greenbug; chinch bug, e.g.,Blissus leucopterus leucopterus; Acrosternum hilare, green stink bug;Euschistus servus, brown stink bug; Jylemya platura, seedcorn maggot;Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite;Oil Seed Rape: Vrevicoryne brassicae, cabbage aphid; Phyllotretacruciferae, crucifer flea beetle; Phyllotreta striolata, striped fleabeetle; Phyllotreta nemorum, striped turnip flea beetle; Meligethesaeneus, rapeseed beetle; and the pollen beetles Meligethes rufimanus,Meligethes nigrescens, Meligethes canadianus, and Meligethesviridescens; Potato: Leptinotarsa decemlineata, Colorado potato beetle.

The methods and compositions provided herein may be effective againstHemiptera such as Lygus hesperus, Lygus lineolaris, Lygus pratensis,Lygus rugulipennis Popp, Lygus pabulinus, Calocoris norvegicus, Orthopscompestris, Plesiocoris rugicollis, Cyrtopeltis modestus, Cyrtopeltisnotatus, Spanagonicus albofasciatus, Diaphnocoris chlorinonis,Labopidicola allii, Pseudatomoscelis seriatus, Adelphocoris rapidus,Poecilocapsus lineatus, Blissus leucopterus, Nysius ericae, Nysiusraphanus, Euschistus servus, Nezara viridula, Eurygaster, Coreidae,Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, and Cimicidae. Pestsof interest also include Araecerus fasciculatus, coffee bean weevil;Acanthoscelides obtectus, bean weevil; Bruchus rufmanus, broadbeanweevil; Bruchus pisorum, pea weevil; Zabrotes subfasciatus, Mexican beanweevil; Diabrotica balteata, banded cucumber beetle; Cerotomatrifurcata, bean leaf beetle; Diabrotica virgifera, Mexican cornrootworm; Epitrix cucumeris, potato flea beetle; Chaetocnema confinis,sweet potato flea beetle; Hypera postica, alfalfa weevil; Anthonomusquadrigibbus, apple curculio; Sternechus paludatus, bean stalk weevil;Hypera brunnipennis, Egyptian alfalfa weevil; Sitophilus granaries,granary weevil; Craponius inaequalis, grape curculio; Sitophiluszeamais, maize weevil; Conotrachelus nenuphar, plum curculio; Euscepespostfaciatus, West Indian sweet potato weevil; Maladera castanea,Asiatic garden beetle; Rhizotrogus majalis, European chafer;Macrodactylus subspinosus, rose chafer; Tribolium confusum, confusedflour beetle; Tenebrio obscurus, dark mealworm; Tribolium castaneum, redflour beetle; Tenebrio molitor, yellow mealworm.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Insect pests may be tested for pesticidal activity of compositions ofthe invention in early developmental stages, e.g., as larvae or otherimmature forms. The insects may be reared in total darkness at fromabout 20 degree C. to about 30 degree C. and from about 30% to about 70%relative humidity. Bioassays may be performed as described in Czapla andLang (1990) J. Econ. Entomol. 83 (6): 2480-2485. See, also theexperimental section herein.

III. Expression Cassettes

Polynucleotides encoding the pesticidal proteins provided herein can beprovided in expression cassettes for expression in an organism ofinterest. The cassette will include 5′ and 3′ regulatory sequencesoperably linked to a polynucleotide encoding a pesticidal polypeptideprovided herein that allows for expression of the polynucleotide. Thecassette may additionally contain at least one additional gene orgenetic element to be cotransformed into the organism. Where additionalgenes or elements are included, the components are operably linked.Alternatively, the additional gene(s) or element(s) can be provided onmultiple expression cassettes. Such an expression cassette is providedwith a plurality of restriction sites and/or recombination sites forinsertion of the polynucleotides to be under the transcriptionalregulation of the regulatory regions. The expression cassette mayadditionally contain a selectable marker gene.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a pesticidal polynucleotide of the invention, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism of interest, i.e., a plant orbacteria. The promoters of the invention are capable of directing ordriving expression of a coding sequence in a host cell. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) may be endogenous or heterologous tothe host cell or to each other. As used herein, “heterologous” inreference to a sequence is a sequence that originates from a foreignspecies, or, if from the same species, is substantially modified fromits native form in composition and/or genomic locus by deliberate humanintervention. As used herein, a chimeric gene comprises a codingsequence operably linked to a transcription initiation region that isheterologous to the coding sequence.

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

Additional regulatory signals include, but are not limited to,transcriptional initiation start sites, operators, activators,enhancers, other regulatory elements, ribosomal binding sites, aninitiation codon, termination signals, and the like. See, for example,U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al.(1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter“Sambrook 11”; Davis et al., eds. (1980) Advanced Bacterial Genetics(Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y., and thereferences cited therein.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, inducible, tissue-preferred, orother promoters for expression in the organism of interest. See, forexample, promoters set forth in WO 99/43838 and in U.S. Pat. Nos.8,575,425; 7,790,846; 8,147,856; 8,586832; 7,772,369; 7,534,939;6,072,050; 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; hereinincorporated by reference.

For expression in plants, constitutive promoters also include CaMV 35Spromoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroyet al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al.(1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) PlantMol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730). Induciblepromoters include those that drive expression of pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol.89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon(1985) Plant Mol. Virol. 4:111-116; and WO 99/43819, herein incorporatedby reference. Promoters that are expressed locally at or near the siteof pathogen infection may also be used (Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977; Chen et al.(1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci.USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz etal. (1989) Plant Cell 1:961-968; Cordero et al. (1992) Physiol. Mol.Plant Path. 41:189-200; U.S. Pat. No. 5,750,386 (nematode-inducible);and the references cited therein).

Wound-inducible promoters may be used in the constructions of theinvention. Such wound-inducible promoters include pin II promoter (Ryan(1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) NatureBiotechnology 14:494-498); wun1 and wun2 (U.S. Pat. No. 5,428,148); win1and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin(McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al.(1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters323:73-76); MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); andthe like, herein incorporated by reference.

Tissue-preferred promoters for use in the invention include those setforth in Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al.(1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. GenGenet. 254(3):337-343; Russell et al. (1997) Transgenic Res.6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341;Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al.(1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant CellPhysiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; andGuevara-Garcia et al. (1993) Plant J. 4(3):495-505.

Leaf-preferred promoters include those set forth in Yamamoto et al.(1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol.105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778;Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci.USA 90(20):9586-9590.

Root-preferred promoters are known and include those in Hire et al.(1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutaminesynthetase gene); Keller and Baumgartner (1991) Plant Cell3(10):1051-1061 (root-specific control element); Sanger et al. (1990)Plant Mol. Biol. 14(3):433-443 (mannopine synthase (MAS) gene ofAgrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22(cytosolic glutamine synthetase (GS)); Bogusz et al. (1990) Plant Cell2(7):633-641; Leach and Aoyagi (1991) Plant Science (Limerick)79(1):69-76 (rolC and rolD); Teeri et al. (1989) EMBO J. 8(2):343-350;Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772 (the VfENOD-GRP3gene promoter); and, Capana et al. (1994) Plant Mol. Biol. 25(4):681-691(rolB promoter). See also U.S. Pat. Nos. 5,837,876; 5,750,386;5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108. Seed-preferred promoters include, but are not limitedto, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps(myo-inositol-1-phosphate synthase) (see WO 00/11177 and U.S. Pat. No.6,225,529). Gamma-zein is an endosperm-specific promoter. Globulin 1(Glb-1) is a representative embryo-specific promoter. For dicots,seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy,shrunken 1, shrunken 2, Globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed.

For expression in a bacterial host, promoters that function in bacteriaare well-known in the art. Such promoters include any of the knowncrystal protein gene promoters, including the promoters of any of thepesticidal proteins of the invention, and promoters specific for B.thuringiensis sigma factors. Alternatively, mutagenized or recombinantcrystal protein-encoding gene promoters may be recombinantly engineeredand used to promote expression of the novel gene segments disclosedherein.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers areknown and any can be used. See, for example, PCT/US2015/066648, filed onDec. 18, 2015, herein incorporated by reference in its entirety, whichdiscloses glufosinate resistance sequences that can be employed asselectable markers.

IV. Methods, Host Cells and Plant Cells

As indicated, DNA constructs comprising nucleotide sequences encodingthe pesticidal proteins or active variants or fragment thereof can beused to transform plants of interest or other organisms of interest.Methods for transformation involve introducing a nucleotide constructinto a plant. By “introducing” is intended to introduce the nucleotideconstruct to the plant or other host cell in such a manner that theconstruct gains access to the interior of a cell of the plant or hostcell. The methods of the invention do not require a particular methodfor introducing a nucleotide construct to a plant or host cell, onlythat the nucleotide construct gains access to the interior of at leastone cell of the plant or the host organism. Methods for introducingnucleotide constructs into plants and other host cells are known in theart including, but not limited to, stable transformation methods,transient transformation methods, and virus-mediated methods.

The methods result in a transformed organisms, such as a plant,including whole plants, as well as plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen).

Transgenic plants” or “transformed plants” or “stably transformed”plants or cells or tissues refers to plants that have incorporated orintegrated a polynucleotide encoding at least one pesticidal polypeptideof the invention. It is recognized that other exogenous or endogenousnucleic acid sequences or DNA fragments may also be incorporated intothe plant cell. Agrobacterium- and biolistic-mediated transformationremain the two predominantly employed approaches. However,transformation may be performed by infection, transfection,microinjection, electroporation, microprojection, biolistics or particlebombardment, electroporation, silica/carbon fibers, ultrasound mediated,PEG mediated, calcium phosphate co-precipitation, polycation DMSOtechnique, DEAE dextran procedure, Agro and viral mediated(Caulimoriviruses, Geminiviruses, RNA plant viruses), liposome mediatedand the like.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Methods for transformation are known in the art andinclude those set forth in U.S. Pat. Nos. 8,575,425; 7,692,068;8,802,934; 7,541,517; each of which is herein incorporated by reference.See, also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858;Jones et al. (2005) Plant Methods 1:5; Rivera et al. (2012) Physics ofLife Reviews 9:308-345; Bartlett et al. (2008) Plant Methods 4:1-12;Bates, G. W. (1999) Methods in Molecular Biology 111:359-366; Binns andThomashow (1988) Annual Reviews in Microbiology 42:575-606; Christou, P.(1992) The Plant Journal 2:275-281; Christou, P. (1995) Euphytica85:13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al.(2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski(1995) Plant Physiology 107:1041-1047; Jones et al. (2005) Plant Methods1:5;

Transformation may result in stable or transient incorporation of thenucleic acid into the cell. “Stable transformation” is intended to meanthat the nucleotide construct introduced into a host cell integratesinto the genome of the host cell and is capable of being inherited bythe progeny thereof. “Transient transformation” is intended to mean thata polynucleotide is introduced into the host cell and does not integrateinto the genome of the host cell.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Nail. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

In specific embodiments, the sequences provide herein can be targeted tospecific cite within the genome of the host cell or plant cell. Suchmethods include, but are not limited to, meganucleases designed againstthe plant genomic sequence of interest (D'Halluin et al. 2013 PlantBiotechnol J); CRISPR-Cas9, TALENs, and other technologies for preciseediting of genomes (Feng, et al. Cell Research 23:1229-1232, 2013,Podevin, et al. Trends Biotechnology, online publication, 2013, Wei etal., J Gen Genomics, 2013, Zhang et al (2013) WO 2013/026740); Cre-loxsite-specific recombination (Dale et al. (1995) Plant J 7:649-659;Lyznik, et al. (2007) Transgenic Plant J 1:1-9; FLP-FRT recombination(Li et al. (2009) Plant Physiol 151:1087-1095); Bxb1-mediatedintegration (Yau et al. Plant J (2011) 701:147-166); zinc-fingermediated integration (Wright et al. (2005) Plant J 44:693-705); Cai etal. (2009) Plant Mol Biol 69:699-709); and homologous recombination(Lieberman-Lazarovich and Levy (2011) Methods Mol Biol 701: 51-65);Puchta (2002) Plant Mol Biol 48:173-182).

The sequence provided herein may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassaya, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Preferably, plantsof the present invention are crop plants (for example, maize, sorghum,wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice,soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced polynucleotides.Further provided is a processed plant product or byproduct that retainsthe sequences disclosed herein, including for example, soymeal.

In another embodiment, the genes encoding the pesticidal proteins can beused to transform insect pathogenic organisms. Such organisms includebaculoviruses, fungi, protozoa, bacteria, and nematodes. Microorganismhosts that are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops ofinterest may be selected. These microorganisms are selected so as to becapable of successfully competing in the particular environment with thewild-type microorganisms, provide for stable maintenance and expressionof the gene expressing the pesticidal protein, and desirably, providefor improved protection of the pesticide from environmental degradationand inactivation.

Such microorganisms include archaea, bacteria, algae, and fungi. Ofparticular interest are microorganisms such as bacteria, e.g., Bacillus,Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces,Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes.Fungi include yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas aeruginosa, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,Clavibacter xyli and Azotobacter vinlandir and phytosphere yeast speciessuch as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus,Kluyveromyces veronae, Aureobasidium pollulans, Bacillus thuringiensis,Escherichia coli, Bacillus subtilis, and the like.

Illustrative prokaryotes, both Gram-negative and gram-positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae.Fungi include Phycomycetes and Ascomycetes, e.g., yeast, such asSaccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such asRhodotorula, Aureobasidium, Sporobolomyces, and the like.

Genes encoding pesticidal proteins can be introduced by means ofelectrotransformation, PEG induced transformation, heat shock,transduction, conjugation, and the like. Specifically, genes encodingthe pesticidal proteins can be cloned into a shuttle vector, forexample, pHT3101 (Lerecius et al. (1989) FEMS Microbiol. Letts. 60:211-218. The shuttle vector pHT3101 containing the coding sequence forthe particular pesticidal protein gene can, for example, be transformedinto the root-colonizing Bacillus by means of electroporation (Lereciuset al. (1989) FEMS Microbiol. Letts. 60: 211-218).

Expression systems can be designed so that pesticidal proteins aresecreted outside the cytoplasm of gram-negative bacteria by fusing anappropriate signal peptide to the amino-terminal end of the pesticidalprotein. Signal peptides recognized by E. coli include the OmpA protein(Ghrayeb et al. (1984) EMBO J, 3: 2437-2442).

Pesticidal proteins and active variants thereof can be fermented in abacterial host and the resulting bacteria processed and used as amicrobial spray in the same manner that Bacillus thuringiensis strainshave been used as insecticidal sprays. In the case of a pesticidalprotein(s) that is secreted from Bacillus, the secretion signal isremoved or mutated using procedures known in the art. Such mutationsand/or deletions prevent secretion of the pesticidal protein(s) into thegrowth medium during the fermentation process. The pesticidal proteinsare retained within the cell, and the cells are then processed to yieldthe encapsulated pesticidal proteins.

Alternatively, the pesticidal proteins are produced by introducingheterologous genes into a cellular host. Expression of the heterologousgene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. These cells are then treated underconditions that prolong the activity of the toxin produced in the cellwhen the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated pesticidal proteins may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample U.S. Pat. No. 6,468,523 and U.S. Publication No. 20050138685,and the references cited therein. In the present invention, atransformed microorganism (which includes whole organisms, cells,spore(s), pesticidal protein(s), pesticidal component(s), pest-impactingcomponent(s), mutant(s), living or dead cells and cell components,including mixtures of living and dead cells and cell components, andincluding broken cells and cell components) or an isolated pesticidalprotein can be formulated with an acceptable carrier into a pesticidalor agricultural composition(s) that is, for example, a suspension, asolution, an emulsion, a dusting powder, a dispersible granule, awettable powder, and an emulsifiable concentrate, an aerosol, animpregnated granule, an adjuvant, a coatable paste, and alsoencapsulations in, for example, polymer substances.

Agricultural compositions may comprise a polypeptide, a recombinogenicpolypeptide or a variant or fragment thereof, as disclosed herein. Theagricultural composition disclosed herein may be applied to theenvironment of a plant or an area of cultivation, or applied to theplant, plant part, plant cell, or seed.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular target pests.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders, or fertilizers. The activeingredients of the present invention are normally applied in the form ofcompositions and can be applied to the crop area, plant, or seed to betreated. For example, the compositions of the present invention may beapplied to grain in preparation for or during storage in a grain bin orsilo, etc. The compositions of the present invention may be appliedsimultaneously or in succession with other compounds. Methods ofapplying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the pesticidal proteins produced by the bacterial strains of thepresent invention include, but are not limited to, foliar application,seed coating, and soil application. The number of applications and therate of application depend on the intensity of infestation by thecorresponding pest.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; a carboxylateof a long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate of dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include but are not limited to inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions of the present invention can be in a suitable form fordirect application or as a concentrate of primary composition thatrequires dilution with a suitable quantity of water or other diluantbefore application. The pesticidal concentration will vary dependingupon the nature of the particular formulation, specifically, whether itis a concentrate or to be used directly. The composition contains 1 to98% of a solid or liquid inert carrier, and 0 to 50% or 0.1 to 50% of asurfactant. These compositions will be administered at the labeled ratefor the commercial product, for example, about 0.01 lb-5.0 lb. per acrewhen in dry form and at about 0.01 pts.-10 pts. per acre when in liquidform.

In a further embodiment, the compositions, as well as the transformedmicroorganisms and pesticidal proteins, provided herein can be treatedprior to formulation to prolong the pesticidal activity when applied tothe environment of a target pest as long as the pretreatment is notdeleterious to the pesticidal activity. Such treatment can be bychemical and/or physical means as long as the treatment does notdeleteriously affect the properties of the composition(s). Examples ofchemical reagents include but are not limited to halogenating agents;aldehydes such as formaldehyde and glutaraldehyde; anti-infectives, suchas zephiran chloride; alcohols, such as isopropanol and ethanol; andhistological fixatives, such as Bouin's fixative and Helly's fixative(see, for example, Humason (1967) Animal Tissue Techniques (W.H. Freemanand Co.).

In one aspect, pests may be killed or reduced in numbers in a given areaby application of the pesticidal proteins provided herein to the area.Alternatively, the pesticidal proteins may be prophylactically appliedto an environmental area to prevent infestation by a susceptible pest.Preferably the pest ingests, or is contacted with, apesticidally-effective amount of the polypeptide. By“pesticidally-effective amount” is intended an amount of the pesticidethat is able to bring about death to at least one pest, or to noticeablyreduce pest growth, feeding, or normal physiological development. Thisamount will vary depending on such factors as, for example, the specifictarget pests to be controlled, the specific environment, location,plant, crop, or agricultural site to be treated, the environmentalconditions, and the method, rate, concentration, stability, and quantityof application of the pesticidally-effective polypeptide composition.The formulations or compositions may also vary with respect to climaticconditions, environmental considerations, and/or frequency ofapplication and/or severity of pest infestation.

The active ingredients are normally applied in the form of compositionsand can be applied to the crop area, plant, or seed to be treated.Methods are therefore provided for providing to a plant, plant cell,seed, plant part or an area of cultivation, an effective amount of theagricultural composition comprising the polypeptide, recombinogenicpolypeptide or an active variant or fragment thereof. By “effectiveamount” is intended an amount of a protein or composition has pesticidalactivity that is sufficient to kill or control the pest or result in anoticeable reduction in pest growth, feeding, or normal physiologicaldevelopment. Such decreases in numbers, pest growth, feeding or normaldevelopment can comprise any statistically significant decrease,including, for example a decrease of about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95% or greater.

For example, the compositions may be applied to grain in preparation foror during storage in a grain bin or silo, etc. The compositions may beapplied simultaneously or in succession with other compounds. Methods ofapplying an active ingredient or an agrochemical composition comprisingat least one of the polypeptides, recombinogenic polypeptides orvariants or fragments thereof as disclosed herein, include but are notlimited to, foliar application, seed coating, and soil application.

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with (or susceptible toinfestation by) a pest against which said polypeptide has pesticidalactivity. In some embodiments, the polypeptide has pesticidal activityagainst a lepidopteran, coleopteran, dipteran, hemipteran, or nematodepest, and said field is infested with a lepidopteran, hemipteran,coleopteran, dipteran, or nematode pest. As defined herein, the “yield”of the plant refers to the quality and/or quantity of biomass producedby the plant. By “biomass” is intended any measured plant product. Anincrease in biomass production is any improvement in the yield of themeasured plant product. Increasing plant yield has several commercialapplications. For example, increasing plant leaf biomass may increasethe yield of leafy vegetables for human or animal consumption.Additionally, increasing leaf biomass can be used to increase productionof plant-derived pharmaceutical or industrial products. An increase inyield can comprise any statistically significant increase including, butnot limited to, at least a 1% increase, at least a 3% increase, at leasta 5% increase, at least a 10% increase, at least a 20% increase, atleast a 30%, at least a 50%, at least a 70%, at least a 100% or agreater increase in yield compared to a plant not expressing thepesticidal sequence. In specific methods, plant yield is increased as aresult of improved pest resistance of a plant expressing a pesticidalprotein disclosed herein. Expression of the pesticidal protein resultsin a reduced ability of a pest to infest or feed.

The plants can also be treated with one or more chemical compositions,including one or more herbicide, insecticides, or fungicides.

Non-limiting embodiments include:

Embodiment 1

An isolated polypeptide having insecticidal activity, comprising:

(a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of sequences set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, and/or 218; or

(b) a polypeptide comprising an amino acid sequence having at least thepercent sequence identity set forth in Table 1 to an amino acid sequenceselected from the group consisting of sequences set forth in SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, and/or 218.

Embodiment 2

The polypeptide of embodiment 1, wherein said polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, or 218.

Embodiment 3

The polypeptide of embodiment 1 or 2, further comprising heterologousamino acid sequences.

Embodiment 4

A composition comprising the polypeptide of any one of embodiments 1-3.

Embodiment 5

A recombinant nucleic acid molecule that encodes the polypeptide of anyone of embodiments 1, 2, or 4, wherein said recombinant nucleic acidmolecule is not the naturally occurring sequence encoding saidpolypeptide.

Embodiment 6

The recombinant nucleic acid of embodiment 5, wherein said nucleic acidmolecule is a synthetic sequence that has been designed for expressionin a plant.

Embodiment 7

The recombinant nucleic acid molecule of embodiments 5 or 6, whereinsaid nucleic acid molecule is operably linked to a promoter capable ofdirecting expression in a plant cell.

Embodiment 8

The recombinant nucleic acid molecule of embodiment 5, wherein saidnucleic acid molecule is operably linked to a promoter capable ofdirecting expression in a bacteria.

Embodiment 9

A host cell that contains the recombinant nucleic acid molecule of anyone of embodiments 5-8.

Embodiment 10

The host cell of embodiment 9, wherein said host cell is a bacterialhost cell.

Embodiment 11

A DNA construct comprising a promoter that drives expression in a plantcell operably linked to a recombinant nucleic acid molecule comprising:

(a) a nucleotide sequence that encodes a polypeptide comprising theamino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, and/or 218; or,

(b) a nucleotide sequence that encodes a polypeptide comprising an aminoacid sequence having at least the percent sequence identity set forth inTable 1 to an amino acid sequence selected from the group consisting ofsequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, and/or 218.

Embodiment 12

The DNA construct of embodiment 11, wherein said nucleotide sequence isa synthetic DNA sequence that has been designed for expression in aplant.

Embodiment 13

A vector comprising the DNA construct of embodiments 11 or 12.

Embodiment 14

A host cell that contains the DNA construct of embodiments 11 or 12 orthe vector of embodiment 13.

Embodiment 15

The host cell of embodiment 14, wherein the host cell is a plant cell.

Embodiment 16

A transgenic plant comprising the host cell of embodiment 15.

Embodiment 17

A composition comprising the host cell of embodiments 9, 10, 14, or 15.

Embodiment 18

The composition of embodiment 17, wherein said composition is selectedfrom the group consisting of a powder, dust, pellet, granule, spray,emulsion, colloid, and solution.

Embodiment 19

The composition of embodiments 16 or 17, wherein said compositioncomprises from about 1% to about 99% by weight of said polypeptide.

Embodiment 20

A method for controlling a pest population comprising contacting saidpopulation with a pesticidal-effective amount of the composition of anyone of embodiments 17-19.

Embodiment 21

A method for killing a pest population comprising contacting saidpopulation with a pesticidal-effective amount of the composition of anyone of embodiments 17-19.

Embodiment 22

A method for producing a polypeptide with pesticidal activity,comprising culturing the host cell of any one of embodiments 9, 10, 14,or 15 under conditions in which the nucleic acid molecule encoding thepolypeptide is expressed.

Embodiment 23

A plant having stably incorporated into its genome a DNA constructcomprising a nucleotide sequence that encodes a protein havingpesticidal activity, said nucleotide sequence comprising:

(a) a nucleotide sequence that encodes a polypeptide comprising theamino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, or 218; or,

(b) a nucleotide sequence that encodes a polypeptide comprising an aminoacid sequence having at least the percent sequence identity set forth inTable 1 to an amino acid sequence selected from the group consisting ofsequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, or 218.

Embodiment 24

A transgenic seed of the plant of embodiment 23.

Embodiment 25

A method for protecting a plant from an insect pest, comprisingexpressing in a plant or cell thereof a nucleotide sequence that encodesa pesticidal polypeptide, said nucleotide sequence comprising:

(a) a nucleotide sequence that encodes a polypeptide comprising theamino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, or 218; or,

(b) a nucleotide sequence that encodes a polypeptide comprising an aminoacid sequence having at least the percent sequence identity set forth inTable 1 to an amino acid sequence selected from the group consisting ofsequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, or 218.

Embodiment 26

The method of embodiment 25, wherein said plant produces a pesticidalpolypeptide having pesticidal against a lepidopteran or coleopteranpest.

Embodiment 27

A method for increasing yield in a plant comprising growing in a field aplant or seed thereof having stably incorporated into its genome a DNAconstruct comprising a promoter that drives expression in a plantoperably linked to a nucleotide sequence that encodes a pesticidalpolypeptide, wherein said nucleotide sequence comprises:

(a) a nucleotide sequence that encodes a polypeptide comprising theamino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, or 218; or,

(b) a nucleotide sequence that encodes a polypeptide comprising an aminoacid sequence having at least the percent sequence identity set forth inTable 1 to an amino acid sequence selected from the group consisting ofsequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, or 218.

Embodiment 28

A method of obtaining a polynucleotide that encodes an improvedpolypeptide comprising pesticidal activity is provided, wherein theimproved polypeptide has at least one improved property over any one ofSEQ ID NOS: 1-218 comprising:

(a) recombining a plurality of parental polynucleotides comprising SEQID NO: 1-218 or an active variant or fragment thereof to produce alibrary of recombinant polynucleotides encoding recombinant pesticidalpolypeptides;

(b) screening the library to identify a recombinant polynucleotide thatencodes an improved recombinant pesticidal polypeptide that has anenhanced property improved over the parental polynucleotide;

(c) recovering the recombinant polynucleotide that encodes the improvedrecombinant pesticidal polypeptide identified in (b); and,

(d) repeating steps (a), (b) and (c) using the recombinantpolynucleotide recovered in step (c) as one of the plurality of parentalpolynucleotides in repeated step (a).

Embodiment 29

An isolated polypeptide having insecticidal activity, comprising: (a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of sequences set forth in SEQ ID NOs: 205, 206, 207, 208,209, 210, 211, 212, 213, or 214, and further comprising at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more of the modifications set forth in Table3; or (b) a polypeptide comprising an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to any one of SEQ ID NOS: 205, 206,207, 208, 209, 210, 211, 212, 213, or 214 and further comprising atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the modifications setforth in Table 3.

Embodiment 30

The polypeptide of embodiment 29, wherein said polypeptide comprises theamino acid sequence set forth in SEQ ID NO:205, 206, 207, 208, 209, 210,211, 212, 213, or 214, and further comprising at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more of the modifications set forth in Table 3.

Embodiment 31

The polypeptide of embodiment 29 or 30, further comprising heterologousamino acid sequences.

Embodiment 32

A composition comprising the polypeptide of any one of embodiments 29,30, or 31.

Embodiment 33

A recombinant nucleic acid molecule that encodes the polypeptide of anyone of embodiments 1 to 3, wherein said recombinant nucleic acidmolecule is not the naturally occurring sequence encoding saidpolypeptide.

Embodiment 34

The recombinant nucleic acid of embodiment 33, wherein said nucleic acidmolecule is a synthetic sequence that has been designed for expressionin a plant.

Embodiment 35

The recombinant nucleic acid molecule of embodiment 33 or 34, whereinsaid nucleic acid molecule is operably linked to a promoter capable ofdirecting expression in a plant cell.

Embodiment 36

The recombinant nucleic acid molecule of any one of embodiments 33, 34,or 35, wherein said nucleic acid molecule is operably linked to apromoter capable of directing expression in a bacteria.

Embodiment 37

A host cell that contains the recombinant nucleic acid molecule of anyone of embodiments 33, 34, 35 or 36.

Embodiment 38

The host cell of embodiment 37, wherein said host cell is a bacterialhost cell.

Embodiment 39

A DNA construct comprising a promoter that drives expression in a plantcell operably linked to a recombinant nucleic acid molecule comprising:(a) a nucleotide sequence that encodes a polypeptide, wherein thepolypeptide comprises the amino acid sequence of any one of SEQ IDNOS:205, 206, 207, 208, 209, 210, 211, 212, 213, or 214, and furthercomprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of themodifications set forth in Table 3; or (b) a nucleotide sequence thatencodes a polypeptide, wherein the polypeptide comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQID NOS: 205, 206, 207, 208, 209, 210, 211, 212, 213, or 214 and furthercomprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of themodifications set forth in Table 3.

Embodiment 40

The DNA construct of embodiment 39, wherein said nucleotide sequence isa synthetic DNA sequence that has been designed for expression in aplant.

Embodiment 41

A vector comprising the DNA construct of embodiment 39 or 40.

Embodiment 42

A host cell that contains the DNA construct of any one of embodiments39, 40 or 41 or the vector of embodiment 41.

Embodiment 43

The host cell of embodiment 42, wherein the host cell is a plant cell.

Embodiment 44

A transgenic plant comprising the host cell of embodiment 42 or 43.

Embodiment 45

A composition comprising the host cell of any one of embodiments 37, 38,42 or 43.

Embodiment 46

The composition of embodiment 45, wherein said composition is selectedfrom the group consisting of a powder, dust, pellet, granule, spray,emulsion, colloid, and solution.

Embodiment 47

The composition of embodiment 45 or 46, wherein said compositioncomprises from about 1% to about 99% by weight of said polypeptide.

Embodiment 48

A method for controlling a pest population comprising contacting saidpopulation with a pesticidal-effective amount of the composition of anyone of embodiments 32 or 45-47.

Embodiment 49

A method for killing a pest population comprising contacting saidpopulation with a pesticidal-effective amount of the composition of anyone of embodiments 32 or 45-47.

Embodiment 50

A method for producing a polypeptide with pesticidal activity,comprising culturing the host cell of any one of embodiments 37, 38, 42,or 43 under conditions in which the nucleic acid molecule encoding thepolypeptide is expressed.

Embodiment 51

A plant having stably incorporated into its genome a DNA constructcomprising a nucleotide sequence that encodes a protein havingpesticidal activity, wherein said nucleotide sequence comprises: (a) anucleotide sequence that encodes a polypeptide, wherein the polypeptidecomprises the amino acid sequence of any one of SEQ ID NOS: 205, 206,207, 208, 209, 210, 211, 212, 213, or 214, and further comprises atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the modifications setforth in Table 3; or (b) a nucleotide sequence that encodes apolypeptide, wherein the polypeptide comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS:205, 206, 207, 208, 209, 210, 211, 212, 213, or 214 and furthercomprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of themodifications set forth in Table 3.

Embodiment 52

A transgenic seed of the plant of embodiment 51.

Embodiment 53

A method for protecting a plant from an insect pest, comprisingexpressing in a plant or cell thereof a nucleotide sequence that encodesa pesticidal polypeptide, wherein said nucleotide sequence comprises:(a) a nucleotide sequence that encodes a polypeptide comprising theamino acid sequence of any one of SEQ ID NOS: 205, 206, 207, 208, 209,210, 211, 212, 213, or 214, and further comprising at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more of the modifications set forth in Table 3; or(b) a nucleotide sequence that encodes a polypeptide comprising an aminoacid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one ofSEQ ID NOS: 205, 206, 207, 208, 209, 210, 211, 212, 213, or 214 andfurther comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ofthe modifications set forth in Table 3.

Embodiment 54

The method of embodiment 53, wherein said plant produces a pesticidalpolypeptide having pesticidal activity against a lepidopteran orcoleopteran pest.

Embodiment 55

A method for increasing yield in a plant comprising growing in a field aplant or seed thereof having stably incorporated into its genome a DNAconstruct comprising a promoter that drives expression in a plantoperably linked to a nucleotide sequence that encodes a pesticidalpolypeptide, wherein said nucleotide sequence comprises: (a) anucleotide sequence that encodes a polypeptide comprising the amino acidsequence of any one of SEQ ID NOS: 205, 206, 207, 208, 209, 210, 211,212, 213, or 214, and further comprising at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more of the modifications set forth in Table 3; or (b) anucleotide sequence that encodes a polypeptide comprising an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ IDNOS: 205, 206, 207, 208, 209, 210, 211, 212, 213, or 214 and furthercomprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of themodifications set forth in Table 3.

Embodiment 56

A method of obtaining a polynucleotide that encodes an improvedpolypeptide comprising pesticidal activity is provided, wherein theimproved polypeptide has at least one improved property over any one ofSEQ ID NOS: 205, 206, 207, 208, 209, 210, 211, 212, 213, or 214comprising: (a) recombining a plurality of parental polynucleotidescomprising SEQ ID NO: 205, 206, 207, 208, 209, 210, 211, 212, 213, or214 or an active variant or fragment thereof to produce a library ofrecombinant polynucleotides encoding recombinant pesticidalpolypeptides; (b) screening the library to identify a recombinantpolynucleotide that encodes an improved recombinant pesticidalpolypeptide that has an enhanced property improved over the parentalpolynucleotide; (c) recovering the recombinant polynucleotide thatencodes the improved recombinant pesticidal polypeptide identified in(b); and (d) repeating steps (a), (b) and (c) using the recombinantpolynucleotide recovered in step (c) as one of the plurality of parentalpolynucleotides in repeated step (a).

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1. Discovery of Novel Genes by Sequencing and DNAAnalysis

Microbial cultures were grown in liquid culture in standard laboratorymedia. Cultures were grown to saturation (16 to 24 hours) before DNApreparation. DNA was extracted from bacterial cells by detergent lysis,followed by binding to a silica matrix and washing with an ethanolbuffer. Purified DNA was eluted from the silica matrix with a mildlyalkaline aqueous buffer.

DNA for sequencing was tested for purity and concentration byspectrophotometry. Sequencing libraries were prepared using the NexteraXT library preparation kit according to the manufacturer's protocol.Sequence data was generated on a HiSeq 2000 according to the IlluminaHiSeq 2000 System User Guide protocol.

Sequencing reads were assembled into draft genomes using the CLC BioAssembly Cell software package. Following assembly, gene calls were madeby several methods and resulting gene sequences were interrogated toidentify novel homologs of pesticidal genes. Novel genes were identifiedby BLAST, by domain composition, and by pairwise alignment versus atarget set of pesticidal genes. A summary of such sequences is set forthin Table 1 and as in SEQ ID NOS: 1-218.

Genes identified in the homology search were amplified from bacterialDNA by PCR and cloned into bacterial expression vectors containing fusedin-frame purification tags. Cloned genes were expressed in E. coli andpurified by column chromatography. Purified proteins were assessed ininsect diet bioassay studies to identify active proteins.

Example 2. Heterologous Expression in E. Coli

The open reading frame set forth in SEQ ID NO: 209 (APG01037.1) wascloned into an E. coli expression vector containing a 6×HIS tag (pHIS).The expression vector was transformed into BL21*RIPL. An LB culturesupplemented with kanyamycin was inoculated with a single colony andgrown overnight at 37° C. using 0.5% of the overnight culture, a freshculture was inoculated and grown to logarithmic phase at 37 degrees C.The culture was induced using 250 mM IPTG for 18 hours at 16° C. Thecells were pelleted and resuspended in 10 mM Tris pH7.4 and 150 mM NaClsupplemented with protease inhibitors. The protein expression wasevaluated by SDS-PAGE.

Example 3. Pesticidal Activity Against Coleopteran and Lepidoptera

Protein Expression:

The sequence set forth in SEQ ID NO: 209 (APG01037.1) was expressed inE. coli as described in Example 2. 400 mL of LB was inoculated and grownto an OD600 of 0.6. The culture was induced with 250 mM IPTG overnightat 16° C. The cells were spun down and the cell pellet was resuspend in5 mL of buffer. The resuspension was bead beaten for 2 min at 4° C.

Bioassay:

Fall army worm (FAW), corn ear worm (CEW), European corn borer (ECB)southwestern corn borer (SWCB) and diamond backed moth (DBM) eggs werepurchased from a commercial insectary (Benzon Research Inc., Carlisle,Pa.). The FAW, CEW, ECB and BCW eggs were incubated to the point thateclosion would occur within 12 hrs of the assay setup. SWCB and DBM wereintroduced to the assay as neonate larvae. Assays were carried out in24-well trays containing multispecies lepidopteran diet (SOUTHLANDPRODUCTS INC., Lake Village, Ark.). Samples of the bead beaten lysatewere applied to the surface of the diet (diet overlay) and allowed toevaporate and soak into the diet. For CEW, FAW, BCW, ECB and SWCB, a 125μl of bead beaten lysate was added to the diet surface and dried. ForDBM, 50 μl of a 1:2 dilution of bead beaten lysate was added to the dietsurface. The bioassay plates were sealed with a plate sealing filmvented with pin holes. The plates were incubated at 26 C at 65% RH on a16:8 day:night cycle in a Percival for 5 days. The assays were assessedfor level of mortality, growth inhibition and feeding inhibition.

For the western corn rootworm bioassay, the protein construct/lysate wasevaluated in an insect bioassay by dispensing 60 μl of a 1:6 dilution ofbead beaten lysate to the top surface of diet in well/s of 24-well plate(Cellstar, 24-well, Greiner Bio One) and allowed to dry. Each wellcontains 500 μl diet (Marrone et al., 1985). Fifteen to twenty neonatelarvae were introduced in each well using a fine tip paint brush and theplate was covered with membrane (Viewseal, Greiner Bio One). Thebioassay was stored at ambient temperature and scored for mortality,and/or growth/feeding inhibition at day 4. FIG. 2 provides the assayscoring guidelines for the corn root worm bioassay.

For Colorado Potato Beetle (CPB) a cork bore size No. 8 leaf disk wasexcised from potato leaf and is dipped in the protein bead beaten lysatewith 0.1% Tween80 until thoroughly wet and placed on top of filter disk(Millipore, glass fiber filter, 13 mm). Sixty μl dH₂O was added to eachfilter disk and placed in each well of 24-well plate (Cellstar, 24-well,Greiner Bio One). The leaf disk was allowed to dry and five to sevenfirst instar larvae were introduced in each well using a fine tip paintbrush. The plate is covered with membrane (Viewseal, Greiner Bio One)and a small hole was punctured in each well of the membrane. Theconstruct was evaluated with four replicates, and scored for mortalityand leaf damage on day 3.

The data from the various Lepidoptera bioassays is set forth in Table 4,and the scoring chart for the Lepidoptera bioassay is found in Table 5.As shown, SEQ ID NO: 209 has pesticidal activity against Lepidoptera.

TABLE 4 Pesticidal activity of SEQ ID NO: 209 (APG01037.1) againstvarious Lepidoptera European Plutella Corn Fall Black Southwestern Cornxylostella Earworm Armyworm Cutworm Corn Borer Borer (Px) (CEW) (FAW)(BCW) (SWCB) (ECB) CrylAc 5 5 3 5 5 5 SEQ ID NO: 209 5 4 3 5 4 5(APG01037.1) MBP empty vector 0 0 1 0 1 0

TABLE 5 Scoring scale for Lepidoptera bioassay 0 no effect 1 slightstunt 2 stunt, low feeding 3 stunt, some mortality, low feeding 4 stunt,some mortality, very low feeding 5 stunt, complete mortality, very lowfeeding

The data from the various Coleopteran bioassays is set forth in Table 6.The CPB assay was run using a leaf disk. The leaf disk was soaked inbead beaten lysate with 0.1% Tween 80 and then CPB was placed on theleaf to look at both mortality and feeding. The more damage to the leaf,the more feeding. APG01037.1 (SEQ ID NO: 209) had 100% mortality with 2%leaf damage in the CPB bioassay. Data not shown. Negative controls(Buffer and empty vector) had 0% mortality with 65% and 70% leaf damagerespectively. This demonstrates APG01037.1 SEQ ID NO: 209 has pesticidalactivity against coleopteran.

Data from the corn root worm bioassay is set forth in Table 6. As shown,APG01037.1 (SEQ ID NO: 209) had 100% mortality and negative controls(Cry1Ac, MBP empty vector and buffer) had less than 10% mortality. Thisdemonstrates APG01037.1 SEQ ID NO: 209 has pesticidal activity againstColeopteran.

TABLE 6 Corn Root Worm Bioassay CRW APG# % Mortality Larva sizeAPG01037.1 100 s, m (SEQ ID NO: 209) Buffer 10 b MBP empty vector 6 bDiet 14 b CRW larva size: big (b), medium (m), and small (s)

Example 4. Pesticidal Activity Against Hemipteran

Protein Expression:

The sequence set forth is SEQ ID NO: 209 was expressed in E. coli asdescribed in Example 2. 400 mL of LB was inoculated and grown to anOD600 of 0.6. The culture was induced with 0.25 mM IPTG overnight at 16C. The cells were spun down and the cell pellet is re-suspend in 5 mL ofbuffer. The resuspension was bead beaten for 2 min on ice.

Second instar SGSB were obtained from a commercial insectary (BenzonResearch Inc., Carlisle, Pa.). A 50% v/v ratio of bead beaten lysatesample to 20% sucrose was employed in the bioassay. Stretched parafilmwas used as a feeding membrane to expose the SGSB to the diet/samplemixture. The plates were incubated at 25 C:21 C, 16:8 day:night cycle at65% RH for 5 days.

Mortality is scored for each sample. The controls (MPB empty vector andbuffer) showed 0% mortality. The sample containing SEQ ID NO: 209(APG01037.1) resulted in 100% mortality of the SGSB.

Example 5. Heterologous Expression in E. Coli

Each open reading frame set forth in SEQ ID NO: 205, 206, 207, 208, 209or active variants or fragments thereof or an open reading frame setforth in Table 1 (SEQ ID NO: 1-204 and SEQ ID NOS: 205-218) or an activevariant or fragment thereof was cloned into an E. coli expression vectorcontaining a 6×HIS tag (pHIS). The expression vector was transformedinto BL21*RIPL. An LB culture supplemented with kanamycin was inoculatedwith a single colony and grown overnight at 37 degrees C. using 0.5% ofthe overnight culture, a fresh culture was inoculated and grown tologarithmic phase at 37 degrees C. The culture was induced using 250 mMIPTG for 18 hours at 16 degrees C. The cells were pelleted andresuspended in 10 mM Tris pH7.4 and 150 mM NaCl supplemented withprotease inhibitors. The protein expression was evaluated by SDS-PAGE.

Example 6. Pesticidal Activity Against Coleopteran and Lepidoptera

Protein Expression:

Each of SEQ ID NO: 205, 206, 207, 208, 209 or active variants orfragments thereof or an open reading frame set forth in sequence setforth in Table 1 (SEQ ID NO: 1-218) is expressed in E. coli as describedin Example 5. 400 mL of LB is inoculated and grown to an OD600 of 0.6.The culture is induced with 0.25 mM IPTG overnight at 16 C. The cellsare spun down and the cell pellet is resuspend in 5 mL of buffer. Theresuspension is bead beaten for 2 min at 4 degrees C.

Bioassay:

Fall army worm (FAW), corn ear worm (CEW), European corn borer (ECB)southwestern corn borer (SWCB) and diamond backed moth (DBM) eggs arepurchased from a commercial insectary (Benzon Research Inc., Carlisle,Pa.). The FAW, CEW, ECB and BCW eggs are incubated to the point thateclosion would occur within 12 hrs of the assay setup. SWCB and DBM areintroduced to the assay as neonate larvae. Assays are carried out in24-well trays containing multispecies lepidopteran diet (SOUTHLANDPRODUCTS INCORPORATED, Lake Village, Ark.). Samples of the sonicatedlysate are applied to the surface of the diet (diet overlay) and allowedto evaporate and soak into the diet. For CEW, FAW, BCW, ECB and SWCB, a125 μl of sonicated lysate is added to the diet surface and dried. ForDBM, 50 μl of a 1:2 dilution of sonicated lysate was added to the dietsurface. The bioassay plates are sealed with a plate sealing film ventedwith pin holes. The plates are incubated at 26 C at 65% RH on a 16:8day:night cycle in a Percival for 5 days. The assays are assessed forlevel of mortality, growth inhibition and feeding inhibition.

For the western corn rootworm bioassay, the protein construct/lysate isevaluated as set forth in Example 3. FIG. 2 provides the assay scoringguidelines for the corn root worm bioassay.

For Colorado Potato Beetle (CPB), the protein construct/lysate isevaluated as set forth in Example 3.

Example 7. Pesticidal Activity Against Hemipteran

Protein Expression:

Each of the sequences of SEQ ID NO: 205, 206, 207, 208, 209 or activevariants or fragments thereof or an open reading frame set forth in setforth in Table 1 (SEQ ID NO: 1-218) is expressed in E. coli as describedin Example 5. 400 mL of LB is inoculated and grown to an OD600 of 0.6.The culture is induced with 250 mM IPTG overnight at 16 C. The cells arespun down and the cell pellet is re-suspend in 5 mL of buffer. Theresuspension is bead beaten for 2 min at 4 degree C.

Second instar SGSB are obtained from a commercial insectary (BenzonResearch Inc., Carlisle, Pa.). A 50% v/v ratio of bead beaten lysatesample to 20% sucrose is employed in the bioassay. Stretched parafilm isused as a feeding membrane to expose the SGSB to the diet/samplemixture. The plates are incubated at 25 C:21 C, 16:8 day:night cycle at65% RH for 5 days.

Mortality is scored for each sample.

Example 8. Transformation of Soybean

DNA constructs comprising SEQ ID NO: 205, 206, 207, 208, 209 or anactive variant of fragment thereof or each of SEQ ID NOS: 1-218 oractive variants or fragments thereof operably linked to a promoteractive in a plant are cloned into transformation vectors and introducedinto Agrobacterium as described in U.S. Provisional Application No.62/094,782, filed Dec. 19, 2015, herein incorporated by reference in itsentirety.

Four days prior to inoculation, several loops of Agrobacterium arestreaked to a fresh plate of YEP* medium supplemented with theappropriate antibiotics** (spectinomycin, chloramphenicol andkanamycin). Bacteria are grown for two days in the dark at 28 C. Aftertwo days, several loops of bacteria are transferred to 3 ml of YEPliquid medium with antibiotics in a 125 ml Erlenmeyer flask. Flasks areplaced on a rotary shaker at 250 RPM at 28 C overnight. One day beforeinoculation, 2-3 ml of the overnight culture were transferred to 125 mlof YEP with antibiotics in a 500 ml Erlenmeyer flask. Flasks are placedon a rotary shaker at 250 RPM at 28 C overnight.

Prior to inoculation, the OD of the bacterial culture is checked at OD620. An OD of 0.8-1.0 indicates that the culture is in log phase. Theculture is centrifuged at 4000 RPM for 10 minutes in Oakridge tubes. Thesupernatant is discarded and the pellet is resuspended in a volume ofSoybean Infection Medium (SI) to achieve the desired OD. The culturesare held with periodic mixing until needed for inoculation.

Two or three days prior to inoculation, soybean seeds are surfacesterilized using chlorine gas. In a fume hood, a petri dish with seedsis place in a bell jar with the lid off. 1.75 ml of 12 N HCl is slowlyadded to 100 ml of bleach in a 250 ml Erlenmeyer flask inside the belljar. The lid is immediately placed on top of the bell jar. Seeds areallowed to sterilize for 14-16 hours (overnight). The top is removedfrom the bell jar and the lid of the petri dish is replaced. The petridish with the surface sterilized is then opened in a laminar flow foraround 30 minutes to disperse any remaining chlorine gas.

Seeds are imbibed with either sterile DI water or soybean infectionmedium (SI) for 1-2 days. Twenty to 30 seeds are covered with liquid ina 100×25 mm petri dish and incubated in the dark at 24 C. Afterimbibition, non-germinating seeds are discarded.

Cotyledonary explants is processed on a sterile paper plate with sterilefilter paper dampened using SI medium employing the methods of U.S. Pat.No. 7,473,822, herein incorporated by reference.

Typically, 16-20 cotyledons are inoculated per treatment. The SI mediumused for holding the explants is discarded and replaced with 25 ml ofAgrobacterium culture (OD 620=0.8-20). After all explants are submerged,the inoculation is carried out for 30 minutes with periodic swirling ofthe dish. After 30 minutes, the Agrobacterium culture is removed.

Co-cultivation plates is prepared by overlaying one piece of sterilepaper onto Soybean Co-cultivation Medium (SCC). Without blotting, theinoculated cotyledons is cultured adaxial side down on the filter paper.Around 20 explants can be cultured on each plate. The plates are sealedwith Parafilm and cultured at 24 C and around 120 umoles m-2s-1 (in aPercival incubator) for 4-5 days.

After co-cultivation, the cotyledons are washed 3 times in 25 ml ofSoybean Wash Medium with 200 mg/l of cefotaxime and timentin. Thecotyledons are blotted on sterile filter paper and then transferred toSoybean Shoot Induction Medium (SSI). The nodal end of the explant isdepressed slightly into the medium with distal end kept above thesurface at about 45 deg. No more than 10 explants are cultured on eachplate. The plates are wrapped with Micropore tape and cultured in thePercival at 24 C and around 120 umoles m-2s-1.

The explants are transferred to fresh SSI medium after 14 days. Emergingshoots from the shoot apex and cotyledonary node are discarded. Shootinduction is continued for another 14 days under the same conditions.

After 4 weeks of shoot induction, the cotyledon is separated from thenodal end and a parallel cut is made underneath the area of shootinduction (shoot pad). The area of the parallel cut is placed on SoybeanShoot Elongation Medium (SSE) and the explants cultured in the Percivalat 24 C and around 120 umoles m-2s-1. This step is repeated every twoweeks for up to 8 weeks as long as shoots continue to elongate.

When shoots reach a length of 2-3 cm, they are transferred to SoybeanRooting Medium (SR) in a Plantcon vessel and incubated under the sameconditions for 2 weeks or until roots reach a length of around 3-4 cm.After this, plants are transferred to soil.

Note, all media mentioned for soybean transformation are found in Paz etal. (2010) Agrobacterium-mediated transformation of soybean and recoveryof transgenic soybean plants; Plant Transformation Facility of IowaState University, which is herein incorporated by reference in itsentirety. (See, agron-www.agron.iastate.edu/ptf/protocol/Soybean.pdf.)

Example 9. Transformation of Maize

Maize ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, such as DN62A5S media (3.98 g/LN6 Salts; 1 mL/L (of 1000.times. Stock) N6 Vitamins; 800 mg/LL-Asparagine; 100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/LCasamino acids; 50 g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D).However, media and salts other than DN62A5S are suitable and are knownin the art. Embryos are incubated overnight at 25 degree C. in the dark.However, it is not necessary per se to incubate the embryos overnight.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for about 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842). DNA constructs designed toexpress the proteins set forth SEQ ID NO: 205, 206, 207, 208, 209 or theproteins set forth in Table 1 (SEQ ID NO: 1-218) in plant cells areaccelerated into plant tissue using an aerosol beam accelerator, usingconditions essentially as described in PCT Publication No. WO/0138514.After beaming, embryos are incubated for about 30 min on osmotic media,and placed onto incubation media overnight at 25 degree C. in the dark.To avoid unduly damaging beamed explants, they are incubated for atleast 24 hours prior to transfer to recovery media. Embryos are thenspread onto recovery period media, for about 5 days, 25 degree C. in thedark, then transferred to a selection media. Explants are incubated inselection media for up to eight weeks, depending on the nature andcharacteristics of the particular selection utilized. After theselection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated by methods known inthe art. The resulting shoots are allowed to root on rooting media, andthe resulting plants are transferred to nursery pots and propagated astransgenic plants.

Example 10. Pesticidal Activity Against Nematodes

A. Heterodera Glycine's (Soybean Cyst Nematode) In-Vitro Assay.

Soybean Cyst Nematodes are dispensed into a 96 well assay plate with atotal volume of 100 uls and 100 J2 per well. The protein of interest asset forth in SEQ ID NO: 205, 206, 207, 208, 209 or active variant orfragments thereof or the sequences set forth in Table 1 (any one of SEQID NOS: 1-218) is dispensed into the wells and held at room temperaturefor assessment. Finally the 96 well plate containing the SCN J2 isanalyzed for motility. Data is reported as % inhibition as compared tothe controls. Hits are defined as greater or equal to 70% inhibition.

B. Heterodera Glycine's (Soybean Cyst Nematode) On-Plant Assay

Soybean plants expressing one or more of SEQ ID NO: 205, 206, 207, 208,209 or active variant or fragments thereof or the sequences set forth inSEQ ID NO: 1-218 or active variant or fragment thereof are generated asdescribed elsewhere herein. A 3-week-old soybean cutting is inoculatedwith 5000 SCN eggs per plant. This infection is held for 70 days andthen harvested for counting of SCN cyst that has developed on the plant.Data is reported as % inhibition as compared to the controls. Hits aredefined as greater or equal to 90% inhibition.

C. Meloidogyne incognita (Root-Knot Nematode) In-Vitro Assay

Root-Knot Nematodes are dispensed into a 96 well assay plate with atotal volume of 100 uls and 100 J2 per well. The protein of interestcomprising any one of SEQ ID NO: 205, 206, 207, 208, 209 or activevariant or fragments thereof or the sequences set forth in SEQ ID NO:1-218 or active variant or fragment thereof is dispensed into the wellsand held at room temperature for assessment. Finally the 96 well platecontaining the RKN J2 is analyzed for motility. Data is reported as %inhibition as compared to the controls. Hits are defined as greater orequal to 70% inhibition.

D. Meloidogyne incognita (Root-Knot Nematode) On-Plant Assay

Soybean plants expressing one or more of SEQ ID NO: 205, 206, 207, 208,209 or active variant or fragments thereof or the sequences set forth inSEQ ID NO: 1-218 or active variants or fragments thereof are generatedas described elsewhere herein. A 3-week-old soybean is inoculated with5000 RKN eggs per plant. This infection is held for 70 days and thenharvested for counting of RKN eggs that have developed in the plant.Data is reported as % inhibition as compared to the controls. Hits aredefined as greater or equal to 90% inhibition.

Example 11. Additional Assays for Pesticidal Activity

The various polypeptides set forth in SEQ ID NO: 205, 206, 207, 208, 209or active variant or fragments thereof or the sequences set forth in SEQID NO: 1-218 or active variant or fragment thereof can be tested to actas a pesticide upon a pest in a number of ways. One such method is toperform a feeding assay. In such a feeding assay, one exposes the pestto a sample containing either compounds to be tested or control samples.Often this is performed by placing the material to be tested, or asuitable dilution of such material, onto a material that the pest willingest, such as an artificial diet. The material to be tested may becomposed of a liquid, solid, or slurry. The material to be tested may beplaced upon the surface and then allowed to dry. Alternatively, thematerial to be tested may be mixed with a molten artificial diet, andthen dispensed into the assay chamber. The assay chamber may be, forexample, a cup, a dish, or a well of a microtiter plate.

Assays for sucking pests (for example aphids) may involve separating thetest material from the insect by a partition, ideally a portion that canbe pierced by the sucking mouth parts of the sucking insect, to allowingestion of the test material. Often the test material is mixed with afeeding stimulant, such as sucrose, to promote ingestion of the testcompound.

Other types of assays can include microinjection of the test materialinto the mouth, or gut of the pest, as well as development of transgenicplants, followed by test of the ability of the pest to feed upon thetransgenic plant. Plant testing may involve isolation of the plant partsnormally consumed, for example, small cages attached to a leaf, orisolation of entire plants in cages containing insects.

Other methods and approaches to assay pests are known in the art, andcan be found, for example in Robertson and Preisler, eds. (1992)Pesticide bioassays with arthropods, CRC, Boca Raton, Fla.Alternatively, assays are commonly described in the journals ArthropodManagement Tests and Journal of Economic Entomology or by discussionwith members of the Entomological Society of America (ESA). Any one ofSEQ ID NO: 205, 206, 207, 208, 209 or active variant or fragmentsthereof or the sequences set forth in SEQ ID NOS: 1-218 or activevariant or fragment thereof can be expressed and employed in an assay asset forth herein.

Example 12. Pesticidal Activity Against Coleopteran and Lepidoptera

Protein Expression: Each sequence set forth in Table 7 was expressed inE. coli as described in Example 2. 400 mL of LB was inoculated and grownto an OD600 of 0.6. The culture was induced with 0.25 mM IPTG overnightat 16° C. The cells were spun down and the cell pellet was resuspend in5 mL of buffer. The resuspension was sonicated for 2 min on ice.

Bioassay: Fall armyworm (FAW), corn earworm (CEW), European corn borer(ECB) southwestern corn borer (SWCB) and diamond backed moth (DBM or Px)bioassays were performed as described in Example 6.

For the western corn rootworm bioassay, the protein construct/lysate wasevaluated as described in Example 3. For Colorado Potato Beetle (CPB),the protein construct/lysate was evaluated as described in Example 3.

Table 7 provides a summary of pesticidal activity against coleopteranand lepidoptera of the various sequences. Table code: “−” indicates noactivity seen; “+” indicates pesticidal activity; “NT” indicates nottested; “S” indicates stunt; “SS” indicates slight stunt; “LF” indicateslow feeding, “M” indicates mortality.

TABLE 7 Summary of Pesticidal Activity against Coleopteran andLepidoptera. APG Seq ID FAW CEW BCW ECB SWCB CPB Px WCR Mortality (%)APG01037.1 209 + + + + + + + 100 APG00623.0 207 + + + + + + + 100APG00556.1 206 + + + + + + + 100 APG01037.4 210 + + + + + + + 100APG01037.5 211 + + + + + + + 100 APG01037.6 212 + + + + + + + 100APG01037.7 213 + + + + + + + 100 APG01037.8 214 + + + + + + + 100

Example 13. Pesticidal Activity Against Hemipteran

Protein Expression: Each of the sequences set forth in Table 8 wasexpressed in E. coli as described in Example 2. 400 mL of LB wasinoculated and grown to an OD600 of 0.6. The culture was induced with0.25 mM IPTG overnight at 16° C. The cells were spun down and the cellpellet was re-suspend in 5 mL of buffer. The resuspension was sonicatedfor 2 min on ice.

Bioassay: Second instar SGSB, brown sting bugs (BSB) and brownmarmorated stink bugs (BMSB) were obtained from ABI's insectary. A 50%v/v ratio of sonicated lysate sample to 20% sucrose solution wasemployed in the bioassay. Stretched parafilm was used as a feedingmembrane to expose the SBs to the diet/sample mixture. The plates wereincubated at 25° C.: 21° C., 16:8 day:night cycle at 65% RH for 7 days.

Mortality was scored for each sample. The results are set forth in Table8. A dashed line indicates no mortality was detected. The negativecontrols (empty vector expressed binding domain and buffer only) bothshowed no mortality (0 stinkbugs out of 4).

TABLE 8 Summary of Pesticidal Activity against Hemipteran SGSB BSB BMSBAPG Seq ID (% mortality) (% mortality) (% mortality) APG01037.1 209 100100 100 APG00623.0 207 100 NT NT APG00556.1 206 100 NT NT APG01037.4 210100 NT NT APG01037.5 211 100 100 100 APG01037.6 212 100 NT NT APG01037.7213 100 NT NT APG01037.8 214 100 NT NT

Example 14. Time Course Assay of APG01037.1 (SEQ ID NO: 209) AgainstSouthern Green Stink Bugs

Twenty-four second instar SGSB were exposed to 250 ppm APG01037.1 (SEQID NO: 209) at 50% v/v ratio of purified protein in 20% sucrose asdescribed in Example 13. Assays were scored for mortality on days 4through 10. At day 7, mortality was 50% higher than the control. FIG. 3provides the results of the time course assay of APG01037.1 (SEQ ID NO:209) against SGSB.

Example 15. Time Course Assay of APG01037.1 (SEQ ID NO: 209) AgainstSoybean Aphids

Five SBA were introduced to each well of a 24-well plate using a paintbrush. A membrane was placed over the well and pushed into place with anorifice reducer. APG01037.1 (SEQ ID NO: 209) was pipetted into eachorifice reducer through the top opening at a rate of 25% (50 μlsample+150 μl artificial diet). Orifice reducers are sealed using abreathe easy membrane and a yellow plate lid is placed on top of theplate. Reproduction, adult mortality, and honeydew production wererecorded on days 1 through 5 post-treatment. The protein was tested at125 ppm and 190 ppm. At day 3, mortality was at least 80% higher thanthe control in both concentrations of the protein. FIG. 4 provides theresults of the time course assay of APG01037.1 against Soybean Aphids.

Example 16. Dose-Response Assay of APG01037.1 (SEQ ID NO: 209) AgainstWestern Corn Rootworm

Approximately 50 WCR neonate larvae were exposed to different doses ofAPG01037.1 (SEQ ID NO: 209) in the bioassay setup as described inExample 12. The assay was scored at day 5 for mortality. Dose effectswere observed. A probit analysis was performed. The LC50 was 52.7 ppm.FIG. 5 provides the concentration-response curve of APG01037.1 (SEQ IDNO: 209) against Western Corn Rootworm.

Example 17. Dose-Response Assay of APG01037.5 (SEQ ID NO: 211) AgainstSouthern Green Stink Bug

Twenty-four second instar SGSB were exposed to different dose ofpurified APG01037.5 (SEQ ID NO: 211) diluted in 20% sucrose (50% v:v)using the assay format described in Example 13. Assays were scored formortality on days 5 through 10. Dose effects were observed. FIG. 6provides the results of the time course assay of APG01037.5 (SEQ ID NO:211) against SGSB.

Example 18. Pesticidal Activity of APG1037.4-.8 (SEQ ID NOS: 210, 211,212, 213, and 214) Against Lygus

Bioassay: Lygus eggs were obtained from Ova the Hill Insectary,Columbia, Mo. The eggs were incubated to the point that eclosion wouldoccur within 12 hrs of the assay setup. Five to seven eggs were placedinto the assay and a 20% v/v ratio of purified protein to diet wasemployed in the bioassay. Stretched parafilm was used as a feedingmembrane to expose the lygus to the sample/diet mixture. The plates wereincubated at 25° C.: 21° C., 16:8 day:night cycle at 65% RH for 5 days.

Mortality was scored for each sample. APG1037.4-8 (SEQ ID NO: 210, 211,212, 213, and 214) had mortality greater than 70% mortality. The resultsare set forth in FIG. 7.

Example 19. Dose-Response Assay of APG1037.5 Against Fall Armyworm

Newly hatched FAW was introduced to purified APG1037.5 (SEQ ID NO: 211)through diet overlay bioassay as set forth in Example 12. Due tocannibalism only 2 larvae were placed in each well for testing. Theassay was scored for mortality, growth inhibition and feedinginhibition. Activity was observed at 300 ppm and greater. The resultsare set forth in Table 9.

TABLE 9 Dose response APG01037.5 (SEQ ID NO: 211) against Fall armywormProtein dose Mortality Stunting (ug/cm{circumflex over ( )}2) %Mortality rating rating 250 0 0 0 300 75 2 1 305 75 2 1 310 67 2 2 315100 2 2 Buffer 0 0 0

Example 20. Pesticidal Activity Against Coleopteran and Lepidoptera

Protein Expression:

The sequence set forth in SEQ ID NO: 1-218 were expressed in E. coli asdescribed in Example 2. 400 mL of LB was inoculated and grown to anOD600 of 0.6. The culture was induced with 250 mM IPTG overnight at 16C. The cells were spun down and the cell pellet was resuspend in 5 mL ofbuffer. The resuspension was bead beaten for 2 min at 4 degrees C.

Bioassay:

Fall army worm (FAW), corn ear worm (CEW), European corn borer (ECB)southwestern corn borer (SWCB) and diamond backed moth (DBM) eggs werepurchased from a commercial insectary (Benzon Research Inc., Carlisle,Pa.). The FAW, CEW, ECB and BCW eggs were incubated to the point thateclosion would occur within 12 hrs of the assay setup. SWCB and DBM wereintroduced to the assay as neonate larvae. Assays were carried out in24-well trays containing multispecies lepidopteran diet (SOUTHLANDPRODUCTS INC., Lake Village, Ark.). Samples of the bead beaten lysatewere applied to the surface of the diet (diet overlay) and allowed toevaporate and soak into the diet. For CEW, FAW, BCW, ECB and SWCB, a 125μl of bead beaten lysate was added to the diet surface and dried. ForDBM, 50 μl of a 1:2 dilution of bead beaten lysate was added to the dietsurface. The bioassay plates were sealed with a plate sealing filmvented with pin holes. The plates were incubated at 26 C at 65% RH on a16:8 day:night cycle in a Percival for 5 days. The assays were assessedfor level of mortality, growth inhibition and feeding inhibition.

For the western corn rootworm bioassay, the protein construct/lysate wasevaluated in an insect bioassay by dispensing 60 μl of a 1:6 dilution ofbead beaten lysate to the top surface of diet in well/s of 24-well plate(Cellstar, 24-well, Greiner Bio One) and allowed to dry. Each wellcontains 500 μl diet (Marrone et al., 1985). Fifteen to twenty neonatelarvae were introduced in each well using a fine tip paint brush and theplate was covered with membrane (Viewseal, Greiner Bio One). Thebioassay was stored at ambient temperature and scored for mortality,and/or growth/feeding inhibition at day 4. FIG. 2 provides the assayscoring guidelines for the corn root worm bioassay.

For Colorado Potato Beetle (CPB) a cork bore size No. 8 leaf disk wasexcised from potato leaf and is dipped in the protein bead beaten lysatewith 0.1% Tween80 until thoroughly wet and placed on top of filter disk(Millipore, glass fiber filter, 13 mm). Sixty μl dH₂O was added to eachfilter disk and placed in each well of 24-well plate (Cellstar, 24-well,Greiner Bio One). The leaf disk was allowed to dry and five to sevenfirst instar larvae were introduced in each well using a fine tip paintbrush. The plate is covered with membrane (Viewseal, Greiner Bio One)and a small hole was punctured in each well of the membrane. Theconstruct was evaluated with four replicates, and scored for mortalityand leaf damage on day 3.

The data from the various Lepidoptera and Coleopteran bioassays is setforth in Table 10, and the scoring chart for the Lepidoptera bioassay isfound in Table 11. As shown, SEQ ID NO: 209 has pesticidal activityagainst Lepidoptera.

TABLE 10 Pesticidal activity of the SEQ ID NOS against variousLepidoptera and Coleopterans. WCR Mortality APG Seq ID FAW CEW BCW ECBSWCB CPB Px (%) APG00524.1 Seq ID 18 M, SS — — NT NT NT NT — APG00606.2Seq ID 52 — — SS NT NT NT NT — APG00785.1 Seq ID 151 M,SS — SS NT NT NTNT — APG00785.2 Seq ID 152 M,SS — SS NT NT NT NT — APG00864.0 Seq ID 174SS — SS NT NT NT NT — APG00607.0 Seq ID 54 M,SS — SS NT NT NT NT —APG00784.1 Seq ID 149 HM,S — SS NT NT NT NT — APG00960.2 Seq ID 181 M,SS— SS NT NT NT NT — APG00608.1 Seq ID 56 SS — — NT NT NT NT — APG00534.1Seq ID 23 SS — SS NT NT NT NT — APG00537.1 Seq ID 29 M,SS — SS NT NT NTNT — APG00786.1 Seq ID 154 HM,S — — NT NT NT NT — APG00536.1 Seq ID 26M,SS — SS NT NT NT NT — APG00536.2 Seq ID 25 HM,S — SS NT NT NT NT —APG00638.0 Seq ID 65 SS SS SS NT NT NT NT — APG00781.0 Seq ID 146 — SSSS NT NT NT NT — APG00528.0 Seq ID 20 SS SS — NT NT NT NT — APG00609.0Seq ID 57 SS SS SS NT NT NT NT — APG00587.1 Seq ID 48 HM,S SS — NT NT NTNT — APG00637.2 Seq ID 63 M,S SS — NT NT NT NT — APG00735.1 Seq ID 145M,SS — SS NT NT NT NT — APG00326.1 Seq ID 2 M,SS — — NT NT NT NT —APG00326.2 Seq ID 3 M,SS — SS NT NT NT NT — APG00383.1 Seq ID 7 SS — —NT NT NT NT — APG00687.1 Seq ID 102 SS SS — NT NT NT NT — APG00657.1 SeqID 80 SS SS — NT NT NT NT — APG00710.2 Seq ID 131 SS — — NT NT NT NT —APG00688.1 Seq ID 105 SS — — NT NT NT NT — APG00805.2 Seq ID 165 SS — SNT NT NT NT — APG00493.1 Seq ID 9 HM,S HM, HM, NT NT +/− NT — S SAPG00659.1 Seq ID 82 HM,S M,S HM, NT NT — NT — S APG00494.1 Seq ID 11 S— SS NT NT NT NT — APG01000.1 Seq ID 197 S — SS NT NT NT NT — APG00939.1Seq ID 50 S — SS NT NT NT NT — APG00661.0 Seq ID 83 S — — NT NT NT NT —APG00513.0 Seq ID 14 S SS SS NT NT NT NT — APG00980.1 Seq ID 187 SS SS —NT NT NT NT — APG00707.1 Seq ID 128 SS — SS NT NT NT NT — APG00495.1 SeqID 13 S SS SS NT NT NT NT — APG00693.2 Seq ID 107 SS — — NT NT NT NT —APG00679.0 Seq ID 99 SS SS SS NT NT NT NT — APG00679.1 Seq ID 100 S S —NT NT NT NT — APG00514.1 Seq ID 16 — — SS NT NT NT NT — APG00729.1 SeqID 143 HM,S — SS NT NT NT NT — APG00706.0 Seq ID 126 M,SS — SS NT NT NTNT — APG00622.0 Seq ID 58 M,SS — M,S NT NT — NT — APG00663.0 Seq ID 87SS — M,SS NT NT — NT — APG00543.0 Seq ID 31 M,SS — — NT NT NT NT —APG00543.1 Seq ID 32 M,SS SS SS NT NT NT NT — APG00705.1 Seq ID 121 M,SS— M,SS NT NT NT NT — APG00705.2 Seq ID 123 M,SS — M,SS NT NT NT NT —APG00705.3 Seq ID 124 SS — NT NT NT NT — APG00705.4 Seq ID 122 M,SS —M,SS NT NT NT NT — APG00703.2 Seq ID 119 M,S SS — NT NT NT NT —APG01028.1 Seq ID 201 — SS — NT NT NT NT — APG01028.2 Seq ID 202 M,SS SS— NT NT NT NT — APG00695.1 Seq ID 111 M,S SS — NT NT NT NT — APG00695.2Seq ID 110 M,SS — — NT NT NT NT — APG00555.1 Seq ID 34 SS — — NT NT NTNT — APG00664.1 Seq ID 90 M,S — — NT NT NT NT — APG00677.0 Seq ID 98 SS— — NT NT NT NT — APG01037.1 Seq ID 209 M,S M,S M,S M, S M, S NT NT80-100 APG00623.0 Seq ID 207 M,SS M,SS M, S M, S HM, S NT NT 80-100APG00556.1 Seq ID 206 M,SS M,S M,SS S M, S NT NT 80-100 APG00624.1 SeqID 61 SS — SS NT NT NT NT — APG00675.0 Seq ID 97 SS SS — NT NT NT NT —APG00649.1 Seq ID 76 — — SS NT NT NT NT — APG00988.0 Seq ID 194 — SS —NT NT NT NT — APG00724.0 Seq ID 137 — — SS NT NT NT NT — APG00724.1 SeqID 138 M,S SS SS NT NT NT NT — APG00701.1 Seq ID 114 — — SS NT NT NT NT— APG00806.1 Seq ID 168 M,SS — — NT NT NT NT — APG00557.0 Seq ID 36 M,S— — NT NT NT NT — APG00557.1 Seq ID 37 SS — — NT NT NT NT — APG00722.0Seq ID 136 HM,S — — NT NT NT NT — APG00648.0 Seq ID 73 SS — — NT NT NTNT — APG00648.1 Seq ID 74 — — SS NT NT NT NT — APG00674.0 Seq ID 96 — SSSS NT NT NT NT — APG00718.0 Seq ID 132 M,S — SS NT NT NT NT — APG00641.1Seq ID 67 SS — — NT NT NT NT — APG00912.1 Seq ID 176 SS — — NT NT NT NT— APG00572.0 Seq ID 46 S — — NT NT NT NT — APG00673.1 Seq ID 95 — — SSNT NT NT NT — APG00802.1 Seq ID 163 M,S SS SS NT NT NT NT — APG00810.1Seq ID 173 S — — NT NT NT NT — APG00644.1 Seq ID 71 SS — — NT NT NT NT —APG00644.2 Seq ID 72 M,S — SS NT NT NT NT — APG01112.0 Seq ID 204 HM,SSS M,SS NT NT NT NT — APG00721.0 Seq ID 133 HM,S SS M,SS NT NT NT NT —APG00721.1 Seq ID 134 M,S SS SS NT NT NT NT — APG00558.1 Seq ID 39 HM,SSS SS NT NT NT NT — APG00558.2 Seq ID 40 S SS SS NT NT NT NT —

TABLE 11 Scoring scale for Lepidoptera and Coleopteran bioassay — noeffect SS slight stunt S Stunt M Mortality HM High morality

Example 21. Pesticidal Activity Against Hemipteran

Protein Expression:

The sequence set forth is SEQ ID NO: 160, 9, 82, 58, 59, 87, 209, 207,and 206 was expressed in E. coli as described in Example 2. 400 mL of LBwas inoculated and grown to an OD600 of 0.6. The culture was inducedwith 0.25 mM IPTG overnight at 16 C. The cells were spun down and thecell pellet is re-suspend in 5 mL of buffer. The resuspension was beadbeaten for 2 min on ice.

Second instar SGSB were obtained from a commercial insectary (BenzonResearch Inc., Carlisle, Pa.). A 50% v/v ratio of bead beaten lysatesample to 20% sucrose was employed in the bioassay. Stretched parafilmwas used as a feeding membrane to expose the SGSB to the diet/samplemixture. The plates were incubated at 25 C:21 C, 16:8 day:night cycle at65% RH for 5 days.

Mortality is scored for each sample. The controls (MPB empty vector andbuffer) showed 0% mortality. The data for SEQ ID NO: 160, 9, 82, 58, 59,87, 209, 207, and 206 is set forth in Table 12.

TABLE 12 Shows the pesticidal activity of the SEQ ID NOS againstHemipteran APG Seq ID Tested against SGSB APG00801.0 Seq ID 160 50%APG00493.1 Seq ID 9 50% APG00659.1 Seq ID 82 75% APG00622.0 Seq ID 5875% APG00622.1 Seq ID 59 50% APG00663.0 Seq ID 87 25% APG01037.1 Seq ID209 100% APG00623.0 Seq ID 207 100% APG00556.1 Seq ID 206 75%

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed is:
 1. A recombinant polypeptide having pesticidalactivity, comprising an amino acid sequence having at least 90% sequenceidentity to the amino acid sequence-set forth in SEQ ID NO:58, whereinsaid polypeptide further comprises a heterologous amino acid sequencechemically linked to said polypeptide.
 2. A composition comprising thepolypeptide of claim
 1. 3. A nucleic acid molecule encoding apolypeptide having at least 90% sequence identity to the amino acidsequence set forth in SEQ ID NO:58, wherein said polypeptide haspesticidal activity, and wherein said nucleic acid molecule is operablylinked to a heterologous promoter.
 4. The nucleic acid molecule of claim3, wherein said nucleic acid molecule is a synthetic sequence designedfor expression in a plant.
 5. The nucleic acid molecule of claim 3,wherein said heterologous promoter is capable of directing expression ina plant cell.
 6. The nucleic acid molecule of claim 3, wherein saidheterologous promoter is capable of directing expression in a bacterialcell.
 7. A host cell comprising the nucleic acid molecule of claim
 3. 8.The host cell of claim 7, wherein said host cell is a bacterial hostcell.
 9. A vector comprising a DNA construct comprising a heterologouspromoter that drives expression in a plant cell operably linked to anucleic acid molecule comprising a nucleotide sequence that encodes apolypeptide comprising an amino acid sequence having at least 90%sequence identity to the amino acid sequence set forth in SEQ ID NO: 58,wherein said polypeptide has pesticidal activity.
 10. The vector ofclaim 9, wherein said nucleotide sequence is a synthetic DNA sequencedesigned for expression in a plant.
 11. A host cell comprising thevector of claim
 9. 12. A composition comprising the host cell of claim11.
 13. A method for controlling a pest population comprising contactingsaid pest population with a pesticidal-effective amount of thecomposition of claim
 2. 14. A method for producing a polypeptide withpesticidal activity comprising culturing the host cell of claim 7 underconditions in which the nucleic acid molecule encoding the polypeptideis expressed.
 15. A plant having stably incorporated into its genome aDNA construct comprising a nucleotide sequence that encodes apolypeptide comprising an amino acid sequence having at least 90%sequence identity to the amino acid sequence set forth in SEQ ID NO: 58,wherein said polypeptide has pesticidal activity.
 16. A transgenic seedof the plant of claim 15, wherein the transgenic seed comprises in itsgenome the DNA construct.
 17. A method for protecting a plant from aninsect pest, comprising expressing in a plant or cell thereof anucleotide sequence that encodes a pesticidal polypeptide, wherein saidpesticidal polypeptide has at least 90% sequence identity to the aminoacid sequence set forth in SEQ ID NO:
 58. 18. The method of claim 17,wherein said pesticidal polypeptide has pesticidal activity against atleast one of a lepidopteran pest, a coleopteran pest, or a hemipteranpest.
 19. A method for increasing yield in a plant comprising growing ina field a plant or seed thereof having stably incorporated into itsgenome a DNA construct comprising a promoter that drives expression in aplant operably linked to a nucleotide sequence that encodes a pesticidalpolypeptide, wherein said nucleotide sequence comprises: (a) anucleotide sequence that encodes a polypeptide comprising the amino acidsequence of SEQ ID NO: 58; or (b) a nucleotide sequence that encodes apolypeptide comprising an amino acid sequence having at least 90%sequence identity to the amino acid set forth in SEQ ID NO:58.
 20. Therecombinant polypeptide of claim 1, wherein the polypeptide comprises anamino acid sequence having at least 95% sequence identity to the aminoacid sequence set forth in SEQ ID NO:
 58. 21. The recombinantpolypeptide of claim 1, wherein the polypeptide comprises the amino acidsequence set forth in SEQ ID NO:58.
 22. The nucleic acid molecule ofclaim 3, wherein the nucleic acid molecule encodes a polypeptidecomprising at least 95% sequence identity to the amino acid sequence setforth in SEQ ID NO:58, wherein said polypeptide has pesticidal activity.23. The nucleic acid molecule of claim 3, wherein the nucleic acidmolecule encodes a polypeptide comprising the amino acid sequence setforth in SEQ ID NO:
 58. 24. The vector of claim 9, wherein thenucleotide sequence encodes a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the amino acidsequence set forth in SEQ ID NO:58.
 25. The DNA construct of claim 9,wherein the nucleic acid molecule comprises a nucleotide sequence thatencodes a polypeptide comprising the amino acid sequence set forth inSEQ ID NO:58.
 26. The plant of claim 15, wherein the DNA constructcomprises a nucleotide sequence that encodes a polypeptide comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence set forth in SEQ ID NO: 58, wherein said polypeptide haspesticidal activity.
 27. The plant of claim 15, wherein the DNAconstruct comprises a nucleotide sequence that encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:58.
 28. The method ofclaim 17, wherein said nucleotide sequence encodes a polypeptidecomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence set forth in SEQ ID NO:
 58. 29. The method ofclaim 17, wherein said nucleotide sequence encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:58.